Charged ion channel blockers and methods for use

ABSTRACT

The invention provides compounds of Formula (I), or pharmaceutically acceptable salts thereof: 
                         
The compounds, compositions, methods and kits of the invention are useful for the treatment of pain, itch, and neurogenic inflammation.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 16/815,426filed on Mar. 11, 2020, which claims the benefit of U.S. ProvisionalApplication Ser. No. 62/816,434 filed Mar. 11, 2019 and U.S. ProvisionalApplication Ser. No. 62/931,590 filed Nov. 6, 2019. The entire contentsof the above applications are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates generally to quaternary ammoniumcompounds, pharmaceutical compositions, and methods useful as selectiveinhibitors of pain, cough, and itch sensing neurons (nociceptors, coughreceptors and pruriceptors) and in the treatment of neurogenicinflammation.

BACKGROUND OF THE INVENTION

The invention features compounds, compositions and methods for theselective inhibition of sensory neurons (nociceptors, cough receptorsand pruriceptors) and the treatment of neurogenic inflammation bytargeting nociceptors with a small molecule drug, while minimizingeffects on non-nociceptive neurons or other types of cells. According tothe method of the invention, small, cationic drug molecules gain accessto the intracellular compartment of sensory neurons via entry throughlarge pore receptor/ion channels that are present in pain-cough- anditch-sensing neurons but to a lesser extent or not at all in other typesof neurons or in other types of tissue.

Local anesthetics such as lidocaine and articaine act by inhibitingvoltage-dependent sodium channels in neurons. These anesthetics blocksodium channels and thereby the excitability of all neurons, not justpain-sensing neurons (nociceptors). Thus, while the goal of topical orregional anesthesia is to block transmission of signals in nociceptorsto prevent pain, administration of local anesthetics also producesunwanted or deleterious effects such as general numbness from block oflow threshold pressure and touch receptors, motor deficits and/orparalysis from block of motor axons and other complications from blockof autonomic fibers. Local anesthetics are relatively hydrophobicmolecules that gain access to their blocking site on the sodium channelby diffusing through the cell membrane. Charged derivatives of thesecompounds, which are not membrane-permeable, have no effect on neuronalsodium channels when applied to the external surface of the nervemembrane but can block sodium channels if somehow introduced inside thecell, for example by diffusion from a micropipette used for whole-cellelectrophysiological recording from isolated neurons. Pain-, cough-, anditch-sensing neurons differ from other types of neurons in expressing(in most cases) the TRPV1 receptor/channel, which is activated bypainful heat or by capsaicin, the pungent ingredient in chili pepper.Other types of channels selectively expressed in various types ofpain-sensing, cough-sensing and itch-sensing (pruriceptor) neuronsinclude but are not limited to TRPV2-4, TRPA1, TRPM8, ASIC and P2X(2/3)channels. It is well established that some cationic small molecules suchas QX-314 are able to enter a cell via passage through activated largepore channels such as TRPV1.

Neuropathic, inflammatory, and nociceptive pain differ in theiretiology, pathophysiology, diagnosis, and treatment. Nociceptive painoccurs in response to the activation of a specific subset of highthreshold peripheral sensory neurons, the nociceptors, by intense ornoxious stimuli. It is generally acute, self-limiting and serves aprotective biological function by acting as a warning of potential oron-going tissue damage. It is typically well-localized. Examples ofnociceptive pain include, but are not limited to, traumatic or surgicalpain, labor pain, sprains, bone fractures, burns, bumps, bruises,injections, dental procedures, skin biopsies, and obstructions.

Inflammatory pain is pain that occurs in the presence of tissue damageor inflammation including postoperative (i.e. pain associated with acuteperioperative pain resulting from inflammation caused by tissue trauma(e.g., surgical incision, dissection, burns) or direct nerve injury(e.g., nerve transection, stretching, or compression)), post-traumaticpain, arthritic pain (rheumatoid; or osteoarthritis (i.e. joint pain andstiffness due to gradual deterioration of the joint cartilage; riskfactors include aging, injury, and obesity; commonly affected joints arethe hand, wrist, neck, knee, hip, and spine), pain and pain associatedwith damage to joints, muscle, and tendons as in axial low back pain(i.e. a prevalent, painful condition affecting the lower portion of theback; common causes include muscle strain, spine fracture, bulging orruptured disc, and arthritis), severe nociceptive pain may transition toinflammatory pain if there is associated tissue injury.

Neuropathic pain is a common type of chronic, non-malignant pain, whichis the result of an injury or malfunction in the peripheral or centralnervous system and serves no protective biological function. It isestimated to affect more than 1.6 million people in the U.S. population.Neuropathic pain has many different etiologies, and may occur, forexample, due to trauma, surgery, herniation of an intervertebral disk,spinal cord injury, diabetes, infection with herpes zoster (shingles),HIV/AIDS, late-stage cancer, amputation (including mastectomy), carpaltunnel syndrome, chronic alcohol use, exposure to radiation, and as anunintended side-effect of neurotoxic treatment agents, such as certainanti-HIV and chemotherapeutic drugs. Peripheral neuropathy is caused bydamages to the peripheral nerves from injury, trauma, prolongedpressure, or inflammation causing numbness and pain in correspondingareas of the body.

Neuropathic pain is frequently described as “burning,” “electric,”“tingling,” or “shooting” in nature. It is often characterized bychronic dynamic allodynia (defined as pain resulting from a movingstimulus that does not ordinarily elicit a painful response, such aslight touch) and hyperalgesia (defined as an increased sensitivity to anormally painful stimulus) and may persist for months or years beyondthe apparent healing of any damaged tissues.

Pain may occur in patients with cancer, which may be due to multiplecauses; inflammation, compression, invasion, metastatic spread into boneor other tissues.

There are some conditions where pain occurs in the absence of a noxiousstimulus, tissue damage or a lesion to the nervous system, calleddysfunctional pain and these include but are not limited tofibromyalgia, tension type headache, and irritable bowel disorders.

Migraine is a headache associated with the activation of sensory fibersinnervating the meninges of the brain.

Itch (pruritus) is a dermatological condition that may be localized andgeneralized and can be associated with skin lesions (rash, atopiceczema, wheals). Itch accompanies many conditions including but notlimited to stress, anxiety, UV radiation from the sun, metabolic andendocrine disorders (e.g., liver or kidney disease, hyperthyroidism),cancers (e.g., lymphoma), reactions to drugs or food, parasitic andfungal infections, allergic reactions, diseases of the blood (e.g.,polycythemia vera), and dermatological conditions. Itch is mediated by asubset of small diameter primary sensory neurons, the pruriceptor, thatshare many features of nociceptor neurons, including but not limited toexpression of TRPV1 channels, and other large pore channels (e.g.TRPV2-4, TRPA1, TRPM8, ASIC and P2X(2/3). Certain itch mediators—such aseicosanoids, histamine, bradykinin, ATP, and various neurotrophins haveendovanilloid functions. Topical capsaicin suppresses histamine-induceditch. Pruriceptors like nociceptors are therefore a suitable target forthis method of delivering ion channel blockers.

Cough is a defensive reflex designed to protect the airway from foreignbodies and to aid in the clearance of luminal debris. This reflex,however, can became aberrant in a number of diseases leading to anon-productive dry cough where hyper- or allo-tussive states exist.Hyper- and allo-tussive states are often chronic in nature lastinggreater than three months and can be manifested in many airway diseasesstates including asthma, COPD, asthma-COPD overlap syndrome (ACOS),interstitial pulmonary fibrosis (IPF) and lung cancer. In addition,inappropriate cough reflexes can be manifested acutely and chronicallyfollowing viral infection. Furthermore, chronic cough can be idiopathicin nature with unknown etiology.

Neurogenic inflammation is a mode of inflammation mediated by theefferent (motor) functions of sensory neurons, in which pro-inflammatorymediator molecules released in the periphery by pain-sensing neurons(nociceptors) both activate a variety of inflammatory pathways in immunecells, and also act on the vascular system to alter blood flow andcapillary permeability.

Neurogenic inflammation contributes to the peripheral inflammationelicited by tissue injury, autoimmune disease, infection, allergy,exposure to irritants in a variety of tissues, and is thought to play animportant role in the pathogenesis of numerous disorders (e.g.,migraine, arthritis, rhinitis, gastritis, colitis, cystitis, andsunburn). One way to reduce neurogenic inflammation is to blockexcitability in nociceptors, thereby preventing the activation ofnociceptor peripheral terminals and the release of pro-inflammatorychemicals.

Despite the development of a variety of therapies for pain, itch, andneurogenic inflammation, there is a need for additional agents.

SUMMARY OF THE INVENTION

The present invention provides compounds represented by Formula (I) thatcan be used to treat or prevent pain, itch, and neurogenic inflammation:

wherein:

Y⁻ is a pharmaceutically acceptable anion;

R^(A), R^(B), and R^(C) are each independently selected from H, D,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, OR^(I), CN, NR^(J)R^(K), NR^(L)C(O)R^(M), S(O)R^(N),S(O)₂R^(N), SO₂R^(O)R^(P), SO₂NR^(Q)R^(R), SO₃R^(S), CO₂R^(T);C(O)R^(U), and C(O)NR^(V)R^(W); (preferably H, F, Cl, or CN and morepreferably H);

each of R^(I), R^(J), R^(K), R^(L), R^(M), R^(N), R^(O), R^(P), R^(Q),R^(R), R^(S), R^(U), R^(V), and R^(W) is independently selected from H,D, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl; R^(J) and R^(K) or R^(V) and R^(W) or R^(Q) and R^(R)can also be taken together with the nitrogen to which they are attachedto form a substituted or unsubstituted 5, 6, 7, or 8 membered ring;

R^(A), R^(B), and/or R^(C) can be taken together with the phenyl ring towhich they are attached can form a fused bicyclic or tricyclic ringsystem, such as naphthyl, dihydroindenyl, tetrahydronaphthyl,quinolinyl, indolyl, and the like;

X¹ is selected from —CR^(X)R^(Y)—, —NR^(Z)C(O)—, —OC(O)—, —SC(O)—,—C(O)NR^(1A)—, —C(O)O—, —C(O)—, —(O)CS—, —NR^(1A)S(O)—, —S(O)NR^(1A)—,—NR^(1A)C(O)NR^(1A)—, —S(O)— and —S(O)₂—; X¹ can also be—NR^(Z)C(O)CR^(X)R^(Y)—;

each of R^(X), R^(Y), R^(Z), and R^(1A) is independently selected fromH, D, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, and substituted orunsubstituted heteroalkyl;

each of R^(D) and R^(E) is independently selected from H, D, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedheteroalkyl, and substituted or unsubstituted cycloalkyl; or R^(D) andR^(E) together with the carbon to which they are attached form asubstituted or unsubstituted 3-6-membered cycloalkyl (a C₃-C₆cycloalkyl), substituted or unsubstituted heterocyclic, or substitutedor unsubstituted heteroalkyl ring;

-   -   or R^(D) and R^(Z) together with the carbon and the —N—C(O)— to        which they are attached form an optionally substituted        5-8-membered lactam;

R^(F) and R^(G) together with N⁺ form an optionally substitutedheterocyclic ring having one or more nitrogen atoms; or, each of R^(F)and R^(G) is independently selected from substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted heteroalkyl, andsubstituted or unsubstituted C₃-6 cycloalkyl; and

R^(H) is a substituted or unsubstituted aryl ring, or a substituted orunsubstituted heteroaryl ring.

In another embodiment, R^(H) can be a substituted alkyl. The substituentis preferably an ester group, such as —OC(O)R^(1B) wherein R^(1B) issubstituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, and substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R^(1B) is preferably asubstituted or unsubstituted phenyl. R^(H) is preferably —CH₂OC(O)—phenyl.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds represented by Formula (I) asdescribed above, or pharmaceutically acceptable salts, stereoisomers,solvates, hydrates or combinations thereof. The invention also providescompositions comprising compounds having Formula (I) or apharmaceutically acceptable salts thereof, for example, a compositioncomprising an effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient. The compositions of the invention may furthercomprise compounds of the invention and a biologically active agent. Thecompositions can be formulated for oral, intravenous, intramuscular,rectal, cutaneous, subcutaneous, topical, transdermal, sublingual,nasal, inhalation, vaginal, intrathecal, epidural, or ocularadministration.

The invention further provides methods for treating pain, cough, itch,or a neurogenic inflammatory disorder in a patient, includingadministering to the patient a composition comprising a compound havingFormula (I), wherein the compound inhibits one or more voltage-gated ionchannels present in nociceptors and/or cough receptors and/orpruriceptors when exposed or applied to the internal face of thechannels but does not substantially inhibit the channels when applied tothe external face of the channels, and wherein the compound is capableof entering nociceptors, cough receptors or pruriceptors through a largepore channel when the channel is activated and inhibiting one or morevoltage-gated ion channels present in the nociceptors cough receptors orpruriceptors.

In certain embodiments, the large pore channel is a transient receptorpotential ion channel (TRP channel). In other embodiments, the TRPchannel is activated by an exogenous or endogenous agonist. In yet otherembodiments, the large pore channel is TRPA1, TRPV1-4, TRPM8, ASIC orP2X. In particular embodiments, the compound is capable of enteringnociceptors, cough receptors or pruriceptors through the TRPA1 TRPV1-4,TRPM8, ASIC or P2X receptor/channel when the receptor/channel isactivated. In yet other embodiments, the compound inhibits voltage-gatedsodium channels. In yet another embodiment, the type of pain treated bythe methods, compositions, and kits of the invention is selected fromthe group consisting of neuropathic pain, inflammatory pain, nociceptivepain, pain due to infections, and procedural pain, or wherein theneurogenic inflammatory disorder is selected from the group consistingof allergic inflammation, asthma, chronic cough, conjunctivitis,rhinitis, psoriasis, inflammatory bowel disease, interstitial cystitis,and atopic dermatitis.

We have identified compounds having Formula (I)

that are capable of passing through open large pore channels that areexpressed on nociceptors and/or cough receptors and/or pruriceptors butnot on motor neurons. Because the ion channel blocking compounds of thepresent invention are positively charged, they are notmembrane-permeable and thus cannot enter cells that do not express largepore channels. Since large pore channels are often more active in tissueconditions associated with pain (such as inflammation) due to release ofendogenous ligands or activation by thermal stimuli, the ion channelblocker of the invention can be used alone to selectively targetactivated nociceptors in order to effectively treat (e.g., eliminate oralleviate) pain, cough, itch, or neurogenic inflammation. The ionchannel blockers of the invention can also be used in combination withone or more exogenous large pore channel agonists to selectively targetnociceptors in order to effectively treat (e.g., eliminate or alleviate)pain, itch, or neurogenic inflammation.

Voltage-dependent ion channels in pain-sensing neurons are currently ofgreat interest in developing drugs to treat pain. Blockingvoltage-dependent sodium channels in pain-sensing neurons can block painsignals by interrupting initiation and transmission of the actionpotential. Moreover, blocking voltage-dependent sodium channels innociceptors can reduce or eliminate neurogenic inflammation bypreventing activation of nociceptor peripheral terminals and the releasethereof pro-inflammatory chemicals.

Heretofore, a limitation in treating with molecules that block sodiumchannels or calcium channels is that the vast majority of suchexternally-applied molecules are hydrophobic and can pass throughmembranes. Because of this, they will enter all cells and thus have noselectivity for affecting only nociceptors.

The inhibitors of the present invention are membrane-impermeable and areonly effective when present inside the nociceptor cell, and thus mustpass through the cell membrane via a channel or receptor, such as largepore channels (e.g., TRPAV1-4, TRPA1, TRPM8, ASIC and P2X(2/3)), inorder to produce an effect. Under normal circumstances, most large porechannels in nociceptors are not active but require a noxious thermal,mechanical, or chemical stimulus to activate them. For example, TRPchannels in nociceptors can be activated by an exogenous TRP ligand(i.e. TRP agonist) such as capsaicin, which opens the TRPV1 channel.Thus, one approach to selectively targeting nociceptors is toco-administer the membrane-impermeable ion channel inhibitor with anexogenous TRP ligand that permits passage of the inhibitor through theTRP channel into the cell. In addition to capsaicin, the exogenous TRPligand can also be another capsaicinoid, mustard oil, or lidocaine. Inanother example, TRP channels may be active in response to exogenousirritant activators such as inhaled acrolein from smoke or chemicalwarfare agents such as tear gas.

Under certain circumstances, large pore channels can be activated in theabsence of exogenous large pore channel agonists/ligands by endogenousinflammatory activators that are generated by tissue damage, infection,autoimmunity, atopy, ischemia, hypoxia, cellular stress, immune cellactivation, immune mediator production, and oxidative stress. Under suchconditions, endogenous molecules (e.g., protons, lipids, and reactiveoxygen species) can activate large pore channels expressed onnociceptors, allowing membrane-impermeable, voltage-gated ion channelblockers to gain access to the inside of the nociceptor through theendogenously-activated large pore channels. Endogenous inflammatoryactivators of large pore channels include, for example, prostaglandins,nitric oxide (NO), peroxide (H₂O₂), cysteine-reactive inflammatorymediators like 4-hydroxynonenal, endogenous alkenyl aldehydes,endocannabinoids, and immune mediators (e.g., interleukin 1 (IL-1),nerve growth factor (NGF), and bradykinin, whose receptors are coupledto large pore channels).

Definitions

As used herein, the words “a” and “an” are meant to include one or moreunless otherwise specified.

By “biologically active” is meant that a molecule, including biologicalmolecules, such as nucleic acids, peptides, polypeptides, and proteins,exerts a biological, physical or chemical effect activity on a protein,enzyme, receptor, ligand, antigen, itself or other molecule. Forexample, a “biologically active” molecule may possess, e.g., enzymaticactivity, protein binding activity, or pharmacological activities.

Biologically active agents that can be used in the methods and kitsdescribed herein include, without limitation, TRP1A receptor agonists,TRPV1-4 receptor agonists, ASIC agonists, TRPM8 agonists, P2X receptoragonists, NSAIDs, glucocorticoids, narcotics, anti-proliferative andimmune modulatory agents, an antibody or antibody fragment, anantibiotic, a polynucleotide, a polypeptide, a protein, an anti-canceragent, a growth factor, and a vaccine.

By “inflammation” is meant any types of inflammation, such those causedby the immune system (immune-mediated inflammation) and by the nervoussystem (neurogenic inflammation), and any symptom of inflammation,including redness, heat, swelling, pain, and/or loss of function.

By “neurogenic inflammation” is meant any type of inflammation mediatedor contributed to by neurons (e.g. nociceptors) or any other componentof the central or peripheral nervous system.

The term “pain” is used herein in the broadest sense and refers to alltypes of pain, including acute and chronic pain, such as nociceptivepain, e.g. somatic pain and visceral pain; inflammatory pain,dysfunctional pain, idiopathic pain, neuropathic pain, e.g., centrallygenerated pain and peripherally generated pain, migraine, and cancerpain.

The term “nociceptive pain” is used to include all pain caused bynoxious stimuli that threaten to or actually injure body tissues,including, without limitation, by a cut, bruise, bone fracture, crushinjury, burn, and the like. Pain receptors for tissue injury(nociceptors) are located mostly in the skin, musculoskeletal system, orinternal organs.

The term “somatic pain” is used to refer to pain arising from bone,joint, muscle, skin, or connective tissue. This type of pain istypically well localized.

The term “visceral pain” is used herein to refer to pain arising fromvisceral organs, such as the respiratory, gastrointestinal tract andpancreas, the urinary tract and reproductive organs. Visceral painincludes pain caused by tumor involvement of the organ capsule. Anothertype of visceral pain, which is typically caused by obstruction ofhollow viscus, is characterized by intermittent cramping and poorlylocalized pain. Visceral pain may be associated with inflammation as incystitis or reflux esophagitis.

The term “inflammatory pain” includes pain associates with activeinflammation that may be caused by trauma, surgery, infection andautoimmune diseases.

The term “neuropathic pain” is used herein to refer to pain originatingfrom abnormal processing of sensory input by the peripheral or centralnervous system consequent on a lesion to these systems.

The term “procedural pain” refers to pain arising from a medical, dentalor surgical procedure wherein the procedure is usually planned orassociated with acute trauma.

The term “itch” is used herein in the broadest sense and refers to alltypes of itching and stinging sensations localized and generalized,acute intermittent and persistent. The itch may be idiopathic, allergic,metabolic, infectious, drug-induced, due to liver, kidney disease, orcancer. “Pruritus” is severe itching.

By “patient” is meant any animal. In one embodiment, the patient is ahuman. Other animals that can be treated using the methods,compositions, and kits of the invention include but are not limited tonon-human primates (e.g., monkeys, gorillas, chimpanzees), domesticatedanimals (e.g., horses, pigs, goats, rabbits, sheep, cattle, llamas), andcompanion animals (e.g., guinea pigs, rats, mice, lizards, snakes, dogs,cats, fish, hamsters, and birds).

Compounds useful in the invention include, but are not limited to, thosedescribed herein in any of their pharmaceutically acceptable forms,including isomers such as diastereomers and enantiomers, salts, esters,amides, thioesters, solvates, and polymorphs thereof, as well as racemicmixtures and pure isomers of the compounds described herein. The term“pharmaceutically acceptable anion” as used herein, refers to theconjugate base of a pharmaceutically acceptable acid. Such acids aredescribed in Stahl, P. H. and Wermuth, C. G. (eds.), Handbook ofPharmaceutical Salts: Properties, Selection and Use, Wiley VCH (2008).Pharmaceutically acceptable acids include, but are not limited to,acetic acid, dichloroacetic acid, adipic acid, alginic acid, L-ascorbicacid, L-aspartic acid, benzenesulfonic acid, 4-acetamidobenzoic acid,benzoic acid, p-bromophenylsulfonic acid, (+)-camphoric acid,(+)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid,carbonic acid, cinnamic acid, cyclamic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonicacid, sulfuric acid, boric acid, citric acid, formic acid, fumaric acid,galactaric acid, gentisic acid, D-glucoheptonic acid, D-gluconic acid,D-glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, hydrochloric acid,hydrobromic acid, hydroiodic acid, isobutyric acid, DL-lactic acid,lactobionic acid, lauric acid, maleic acid, (−)-L-malic acid, malonicacid, DL-mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonicacid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinicacid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid,pamoic acid, phosphoric acid, propionic acid, (−)-L-pyroglutamic acid,salicyclic acid, 4-aminosalicyclic acid, sebacic acid, stearic acid,succinic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonicacid, and undecylenic acid. Pharmaceutically acceptable anions includethe conjugate base of any the acids set forth above.

The term “pharmaceutically acceptable salt” represents those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. The salts can beprepared in situ during the final isolation and purification of thecompounds of the invention, or separately by reacting the free basefunction with a suitable organic acid. Representative acid additionsalts include, but are not limited to acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate,lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,malonate, mesylate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,toluenesulfonate, undecanoate, valerate salts, and the like.

In the generic descriptions of compounds of this invention, the numberof atoms of a particular type in a substituent group is generally givenas a range, e.g., an alkyl group containing from 1 to 4 carbon atoms orC₁₋₄ alkyl of C₁-C₄ alkyl. Reference to such a range is intended toinclude specific references to groups having each of the integer numberof atoms within the specified range. For example, an alkyl group from 1to 4 carbon atoms includes each of C₁, C₂, C₃, and C₄ alkyls. Othernumbers of atoms and other types of atoms may be indicated in a similarmanner.

“D” is deuterium.

As used herein, the terms “alkyl” and the prefix “alk-” are inclusive ofboth straight chain and branched chain groups and of cyclic groups,i.e., cycloalkyl. Cyclic groups can be monocyclic or polycyclic andpreferably have from 3 to 6 ring carbon atoms or 3 to 7 carbon atoms,inclusive. Exemplary cyclic groups include cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl groups.

By “C₁₋₄ alkyl” or “C₁-C₄ alkyl” is meant a branched or unbranchedhydrocarbon group having from 1 to 4 carbon atoms. Similarly, a “C₁₋₆alkyl” or “C₁-C₆” is a branched or unbranched hydrocarbon group havingfrom 1 to 6 carbon atoms. An alkyl, including, for example, a C₁₋₄ alkylor C₁₋₆ alkyl group may be substituted or unsubstituted. Exemplarysubstituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio,halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, alkylamino,disubstituted amino, quaternary amino, alkylcarboxy, and carboxylgroups. Exemplary substituents also include alkoxy, aryloxy, sulfhydryl,alkylthio, arylthio, halide (F, Cl, Br or I), hydroxyl, fluoroalkyl,perfluoralkyl, oxo, amino, alkylamino, disubstituted amino, quaternaryamino, amido, ester, alkylcarboxy, alkoxycarbonyl, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl, andureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, aryl, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. C₁₋₄ alkyls include,without limitation, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, andcyclobutyl. C₁₋₆ alkyls include, without limitation, methyl, ethyl,n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclobutyl, cyclopentyl, andcyclohexyl.

An example of a substituted alkyl is a heteroalkyl. By “heteroalkyl” ismeant a branched or unbranched alkyl, cycloalkyl, alkenyl, or alkynylgroup having from 1 to 7 or more carbon atoms in addition to 1, 2, 3 or4 heteroatoms independently selected from the group consisting of N, O,and S. By “C₁₋₇ heteroalkyl” is meant a branched or unbranched alkyl,alkenyl, or alkynyl group having from 1 to 7 carbon atoms in addition to1, 2, 3 or 4 heteroatoms independently selected from the groupconsisting of N, O, S, and P. Heteroalkyls can include, withoutlimitation, tertiary amines, secondary amines, ethers, thioethers,amides, thioamides, carbamates, thiocarbamates, hydrazones, imines,phosphodiesters, phosphoramidates, sulfonamides, and disulfides. Aheteroalkyl may optionally include monocyclic, bicyclic, or tricyclicrings, in which each ring desirably has three to six members. Theheteroalkyl group may be substituted or unsubstituted. Exemplarysubstituents include alkyl, alkoxy, aryloxy, sulfhydryl, alkylthio,arylthio, halide (F, Cl, Br or I), hydroxyl, fluoroalkyl, perfluoralkyl,oxo, amino, alkylamino, disubstituted amino, quaternary amino, amido,ester, alkylcarboxy, alkoxycarbonyl, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl, andureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, aryl, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Examples of C₁₋₇heteroalkyls include, without limitation, methoxymethyl and ethoxyethyl.

An alkenyl is a branched or unbranched hydrocarbon group containing oneor more double bonds. For example, by “C₂₋₆ alkenyl” or “C₂-C₆ alkenyl”is meant a branched or unbranched hydrocarbon group containing one ormore double bonds and having from 2 to 6 carbon atoms. An alkenyl mayoptionally include monocyclic or polycyclic rings, in which each ringdesirably has from three to six members. The alkenyl group may besubstituted or unsubstituted. Exemplary substituents include thosedescribed above for alkyl, and specifically include alkoxy, aryloxy,sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl,perfluoralkyl, amino, alkylamino, disubstituted amino, quaternary amino,alkylcarboxy, and carboxyl groups. C₂₋₆ alkenyls include, withoutlimitation, vinyl, allyl, 2-cyclopropyl-1-ethenyl, 1-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, and2-methyl-2-propenyl.

An alkynyl is a branched or unbranched hydrocarbon group containing oneor more triple bonds. For example, by “C₂₋₆ alkynyl” or “C₂-C₆ alkynyl”is meant a branched or unbranched hydrocarbon group containing one ormore triple bonds and having from 2 to 6 carbon atoms. An alkynyl mayoptionally include monocyclic, bicyclic, or tricyclic rings, in whicheach ring desirably has five or six members. The alkynyl group may besubstituted or unsubstituted. Exemplary substituents those describedabove for alkyl, and specifically include alkoxy, aryloxy, sulfhydryl,alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino,alkylamino, disubstituted amino, quaternary amino, alkylcarboxy, andcarboxyl groups. C₂₋₆ alkynyls include, without limitation, ethynyl,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.

By “heterocyclyl,” “heterocyclic,” or “heterocycloalkyl” is meant astable monocyclic or polycyclic (including a bicyclic or a tricyclic)heterocyclic ring which is saturated, partially unsaturated orunsaturated (including heteroaryl or aromatic), and which consists of 2or more carbon atoms and 1, 2, 3 4 or more heteroatoms independentlyselected from N, O, and S and including any bicyclic or polycyclic groupin which any of the above-defined heterocyclic rings is fused to abenzene ring, heteroaryl, cycloalkyl or heterocycloalkyl. In certainaspects, the heterocyclyl is a 3- to 15-membered ring system, a 3- to12-membered ring system, or a 3- to 9-membered ring system. By “C₂₋₆heterocyclyl” is meant a stable 5- to 7-membered monocyclic or 7- to14-membered bicyclic heterocyclic ring which is saturated, partiallyunsaturated or unsaturated (including heteroaryl or aromatic), and whichconsists of 2 to 6 carbon atoms and 1, 2, 3 or 4 heteroatomsindependently selected from N, O, and S and including any bicyclic groupin which any of the above-defined heterocyclic rings is fused to abenzene ring, heteroaryl, cycloalkyl or heterocycloalkyl. Theheterocyclyl or heteroaryl group may be substituted or unsubstituted.Exemplary substituents include substituted or unsubstituted alkyl, aryl,cycloalkyl, heterocycloalkyl, heteroaryl, alkoxy, aryloxy, sulfhydryl,alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino,alkylamino, disubstituted amino, quaternary amino, alkylcarboxy, oxo,and carboxyl groups. The nitrogen and sulfur heteroatoms may optionallybe oxidized. The heterocyclic ring may be covalently attached via anyheteroatom or carbon atom which results in a stable structure, e.g., animidazolinyl ring may be linked at either of the ring-carbon atompositions or at the nitrogen atom. A nitrogen atom in the heterocyclecan be quaternized. Preferably when the total number of S and O atoms inthe heterocycle exceeds 1, then these heteroatoms are not adjacent toone another. Heterocycles include, without limitation, 1H-indazole,2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,carbazolyl, 4aH-carbazolyl, b-carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinylperimidinyl,phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl,pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, xanthenyl, β-lactam, γ-lactam and δ-lactam. Preferred 5to 10 membered heterocycles include, but are not limited to, pyridinyl,pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl,pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, benzofuranyl,benzothiofuranyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl,isoxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl,benzoxazolinyl, quinolinyl, and isoquinolinyl. Preferred 5 to 6 memberedheterocycles include, without limitation, pyridinyl, quinolinyl,pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl,piperazinyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl,and tetrazolyl. Preferred substituents include phenyl, methyl, ethyl,propyl, butyl, chloro, bromo, fluoro, iodo and oxo.

By “aryl” is meant an aromatic group having a ring system comprised ofcarbon atoms with conjugated π electrons (e.g., phenyl). A “C₆-C₁₂ aryl”or “C₆-C₁₀ aryl” is an aryl group that has from 6 to 12 carbon atoms or6 to 10 carbon atoms, respectively. Aryl groups may optionally includemonocyclic, bicyclic, or tricyclic rings, in which each ring desirablyhas five or six members. A bicyclic or tricyclic ring system can befused (e.g., naphthyl) or not (e.g., biphenyl). The aryl group may besubstituted or unsubstituted. Exemplary substituents include substitutedor unsubstituted alkyl, hydroxyl, alkoxy, aryloxy, sulfhydryl,alkylthio, arylthio, halide, fluoroalkyl, carboxyl, alkylcarboxy, amino,alkylamino, monosubstituted amino, disubstituted amino, and quaternaryamino groups. A preferred aryl group is phenyl.

By “aralkyl” is meant a substituted or unsubstituted alkyl that issubstituted by a substituted or unsubstituted aryl (including, forexample, (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl).

By “C₇₋₁₄ aralkyl” is meant an alkyl substituted by an aryl group (e.g.,benzyl, phenethyl, or 3,4-dichlorophenethyl) having from 7 to 14 carbonatoms.

By “C₃₋₁₀ heterocycloalkyl” is meant an alkyl substituted heterocyclicgroup having from 3 to 10 carbon atoms in addition to one or moreheteroatoms (e.g., 3-furanylmethyl, 2-furanylmethyl,3-tetrahydrofuranylmethyl, or 2-tetrahydrofuranylmethyl).

By “halide” or “halogen” is meant bromine, chlorine, iodine, orfluorine.

By “fluoroalkyl” is meant an alkyl group that is substituted with afluorine atom.

By “alkylcarboxy” is meant a chemical moiety with the formula —(R)—COOH,wherein R is selected from C₁₋₇ alkyl, C₂₋₇ alkenyl, C₂₋₇ alkynyl, C₂₋₆heterocyclyl, C₆₋₁₂ aryl, C₇-14 aralkyl, C₃₋₁₀ heterocycloalkyl, or C₁₋₇heteroalkyl.

By “alkoxy” is meant a chemical substituent of the formula —OR, whereinR is a substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, or substituted or unsubstituted alkynyl or R can be selectedfrom C₁₋₇ alkyl, C₂₋₇ alkenyl, C₂₋₇ alkynyl, C₂₋₆ heterocyclyl, C₆₋₁₂aryl, C₇₋₁₄ aralkyl, C₃₋₁₀ heterocycloalkyl, or C₁₋₇ heteroalkyl.

By “aryloxy” is meant a chemical substituent of the formula —OR, whereinR is a C₆₋₁₂ aryl group. By “alkylthio” is meant a chemical substituentof the formula —SR, wherein R is selected from C₁₋₇ alkyl, C₂₋₇ alkenyl,C₂₋₇ alkynyl, C₂₋₆ heterocyclyl, C₆₋₁₂ aryl, C₇₋₁₄ aralkyl, C₃₋₁₀heterocycloalkyl, or C₁₋₇ heteroalkyl.

By “arylthio” is meant a chemical substituent of the formula —SR,wherein R is a C₆₋₁₂ aryl group.

By “charged moiety” is meant a moiety which gains a proton atphysiological pH thereby becoming positively charged (e.g., ammonium,guanidinium, or amidinium) or a moiety that includes a net formalpositive charge without protonation (e.g., quaternary ammonium). Thecharged moiety may be either permanently charged or transiently charged.

By “therapeutically effective amount” or “effective amount” means anamount sufficient to produce a desired result, for example, thereduction or elimination of pain, cough, itch, or neurogenicinflammation in a patient (e.g., a human) suffering from a condition,disease, or illness that is caused wholly or in part by neurogenicinflammation (e.g. asthma, arthritis, colitis, contact dermatitis,diabetes, eczema, cystitis, chronic refractory cough, post-viral cough,gastritis, migraine headache, psoriasis, rhinitis, rosacea, or sunburn).

“Solvates” means solvent addition forms that contain eitherstoichiometric or nonstoichiometric amounts of solvent.

The compounds of the present invention, including salts of thecompounds, can exist in unsolvated forms as well as solvated forms,including hydrated forms and unhydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope of the present invention. Nonlimiting examples of hydrates includemonohydrates, dihydrates, hemihydrates, etc. In certain aspects, thecompound is a hemihydrate. Nonlimiting examples of solvates includeethanol solvates, acetone solvates, etc.

The compounds of the invention may exist in multiple crystalline oramorphous forms. In general, all physical forms are equivalent for usescontemplated by the present invention and are intended to be within thescope of the invention.

Compounds that can be used in the compositions, kits, and methods of theinvention include compounds having Formula (I), or a pharmaceuticallyacceptable salt thereof:

wherein:

Y⁻ is a pharmaceutically acceptable anion;

R^(A), R^(B), and R^(C) are each independently selected from H, D,halogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, OR^(I),NR^(J)R^(K), NR^(L)C(O)R^(M), S(O)R^(N), S(O)₂R^(N), SO₂R^(O)R^(P),SO₂NR^(Q)R^(R), SO₃R^(S), CO₂R^(T), C(O)R^(U), and C(O)NR^(V)R^(W);

each of R^(I), R^(J), R^(K), R^(L), R^(M), R^(N), R^(O), R^(P), R^(Q),R^(R), R^(S), R^(T), R^(U), R^(V), and R^(W) is independently selectedfrom H, D, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, and substituted or unsubstituted alkynyl;

X¹ is selected from —CR^(X)R^(Y)—, —NR^(Z)C(O)—,—NR^(Z)C(O)CR^(X)R^(Y)—, —OC(O)—, —SC(O)—, —C(O)NR^(1A)—, —C(O)O—,—C(O)—, —(O)CS—, —NR^(1A)S(O)—, —S(O)NR^(1A)—, —NR^(1A)C(O)NR^(1A)—,—S(O)— and —S(O)₂—;

each of R^(X), R^(Y), R^(Z), and R^(1A) is independently selected fromH, D, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, and substituted or unsubstituted alkynyl;

each of R^(D) and R^(E) is independently selected from H, D, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, and substituted or unsubstitutedcycloalkyl; or R^(D) and R^(E) together with the carbon to which theyare attached form a substituted or unsubstituted C₃-C₆ cycloalkyl or asubstituted or unsubstituted heterocyclic (for example, a 5- to7-membered heterocyclic ring);

R^(F) and R^(G) together with the N⁺ to which they are attached form anoptionally substituted heterocyclic ring having zero, one or morenitrogen atoms in addition to the N⁺; or, each of R^(F) and R^(G) isindependently selected from substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted heterocyclyl, and substituted orunsubstituted C₃₋₆ cycloalkyl; and

R^(H) is a substituted or unsubstituted aryl, or a substituted orunsubstituted heteroaryl.

In a preferred embodiment, R^(H) is independently selected from asubstituted or unsubstituted C₅₋₁₀ aryl or a substituted orunsubstituted C₅₋₁₀ heteroaryl.

In a further preferred embodiment, R^(H) is independently selected froma substituted or unsubstituted C₆₋₁₀ aryl or a substituted orunsubstituted 5- to 10-membered heteroaryl.

In some embodiments, R^(H) is a substituted C₅₋₁₀ aryl or a substitutedC₅₋₁₀ heteroaryl optionally substituted with C₁₋₆ alkane, C₁₋₆heteroalkane, carbocycle, substituted carbocycle, heterocarbocycle,substituted heterocarbocycle, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, carboxamide,hydroxyl, ether, amide, ester, sulfonamide, sulfone, amino, amino alkyl,urea, nitrile, or halogen. In a preferred embodiment, the C₁₋₆ alkane isselected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl. In a preferredembodiment, the C₁₋₆ heteroalkane is selected from —O-methyl, —O-ethyl,—O-propyl, —O-isopropyl, —O-butyl, —O-isobutyl, —O— cyclohexyl,—O-cyclopentyl, and -ethyl-O-methyl. In a preferred embodiment, thecarbocycle is selected from cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl. In a preferred embodiment, the heterocarbocycle is selectedfrom aziridine, azetidine, furan, pyrrolidine, pyran, piperidine,piperazine, azepine, and diazapine.

In yet another preferred embodiment, R^(H) is a substituted orunsubstituted C₆₋₁₀ aryl or a substituted or unsubstituted 5- to10-membered heteroaryl. In certain aspects, R^(H) is an unsubstitutedC₆₋₁₀ aryl or an unsubstituted 5- to 10-membered heteroaryl. Inadditional aspects, R^(H) is a C₆₋₁₀ aryl or a 5- to 10-memberedheteroaryl, each optionally substituted with a substituted orunsubstituted C₁-C₆ alkyl, halo, nitrile, hydroxyl, and alkoxy. In yetadditional aspects, R^(H) is a C₆₋₁₀ aryl or a 5- to 10-memberedheteroaryl, each optionally substituted with a substituted orunsubstituted C₁-C₆ alkyl, halo, nitrile, and OR^(2B), wherein R^(2B) ishydrogen or substituted or unsubstituted C₁-C₆ alkyl. In a furtherpreferred embodiment, R^(H) is an unsubstituted phenyl. In additionalembodiments, R^(H) is phenyl substituted with a substituent selectedfrom the group consisting of substituted or unsubstituted C₁-C₆ alkyl,halo, nitrile, hydroxyl, and alkoxy. In additional aspects, R^(H) isphenyl is substituted with a substituent selected from the groupconsisting of substituted or unsubstituted C₁-C₆ alkyl, halo, nitrile,and OR^(2B), wherein R^(2B) is hydrogen or substituted, andunsubstituted C₁-C₆ alkyl. In yet additional embodiments, R^(H) isphenyl substituted with an unsubstituted C₁-C₆ alkyl, halo, nitrile,hydroxyl, or alkoxy. In further aspects, R^(H) is phenyl substitutedwith a substituent selected from the group consisting of unsubstitutedC₁-C₆ alkyl, halo, nitrile, and OR^(I), wherein R^(2B) is hydrogen orsubstituted or unsubstituted C₁-C₆ alkyl.

In yet further aspects, R_(H) is selected from the Z groups shown inTables 1 to 3.

In a preferred embodiment, X¹ is —NHC(O)— or —C(O)NH—. In anotherpreferred embodiment, X¹ is —NHC(O)—.

In preferred embodiments, R^(A), R^(B), and R^(C) are each independentlyselected from H, D, halogen, substituted or unsubstituted C₁₋₄ alkyl,and NR^(J)R^(K); and each of R^(J) and R^(K) is independently selectedfrom H and substituted or unsubstituted C₁₋₄ alkyl.

In a preferred embodiment, each of R^(A) and R^(B) is CH₃, and R^(C) isH, CH₃, halogen, nitrile, methoxy, or ethoxy.

In yet additional preferred aspects, R^(A), R^(B), and R^(C) areindependently selected from H, D, halogen, OR^(I), substituted orunsubstituted C₁-C₄ alkyl, and NR^(J)R^(K); wherein each of R^(I), R^(J)and R^(K) is independently selected from H and substituted orunsubstituted C₁-C₄ alkyl. In a preferred embodiment, each of R^(A) andR^(B) is CH₃, and R^(C) is selected from the group consisting of H, CH₃,halogen, nitrile (cyano), methoxy, and ethoxy. In further preferredembodiments, each of R^(A) and R^(B) is CH₃, and R^(C) is selected fromthe group consisting of H, CH₃, fluoro, chloro, nitrile, methoxy, andethoxy. In additional preferred embodiments, each of R^(A) and R^(B) areCH₃ and R^(C) is hydrogen.

In certain other embodiments, R^(D) is C₁₋₄ alkyl optionally substitutedwith a substituent selected from the group consisting of halogen,oxygen, C₃₋₈ cycloalkyl, aryl, or heteroaryl, and/or R^(E) is H or C₁₋₄alkyl optionally substituted with a substituent selected from the groupconsisting of halogen, oxygen, C₃₋₈ cycloalkyl, aryl, or heteroaryl.

In preferred embodiments each of R^(D) and R^(E) is independentlyselected from H, D, CH₃, CH₂CH₃, (CH₂)₂CH₃, and (CH₂)₃CH₃. In a morepreferred embodiment, R^(E) is hydrogen and R^(D) is CH₃, CH₂CH₃,(CH₂)₂CH₃, or (CH₂)₃CH₃. In certain, other preferred embodiments R^(D)and R^(E) together form a substituted or unsubstituted C₃-C₆ cycloalkyl.

In certain preferred embodiments, R^(D) and R^(E) are both hydrogen. Inyet additional preferred embodiments, R^(D) is hydrogen and R^(E) is analkyl, for example, a C₁-C₆ alkyl or a C₁-C₄ alkyl including, but notlimited to, methyl, ethyl, propyl and butyl. In certain additionalpreferred embodiments, R^(D) and R^(E) are taken together with thecarbon to which they are attached to form a C₃-C₆ cycloalkyl including,but not limited to, cyclopropyl or cyclobutyl.

In preferred embodiments, R^(F) and R^(G) together with the N⁺ to whichthey are attached form a five, six, seven, or eight memberedheterocyclic ring, each optionally substituted, resulting in a compoundof Formula Ia:

Wherein each variable is as defined above, including preferred oralternative embodiments, n is 1, 2, 3, 4 or 5; and R^(1B) is H or asubstituent, such as H, D, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, OR^(1I), CN, NR^(1J)R^(1K),NR^(1L)C(O)R^(1M), S(O)R^(1N), S(O)₂R^(1N), SO₂R^(1O)R^(1P),SO₂NR^(1Q)R^(1R), SO₃R^(1S), CO₂R^(1T), C(O)R^(1U), andC(O)NR^(1V)R^(1W); and wherein each of R^(1I), R^(1J), R^(1K), R^(1L),R^(1M), R^(1N), R^(1O), R^(1P), R^(1Q), R^(1R), R^(1S), R^(1T), R^(1U),R^(1V), and R^(1W) is independently selected from H, D, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl. R^(1J) and R^(1K) orR^(1V) and R^(1W) or R^(1Q) and R^(1R) can also be taken together withthe nitrogen to which they are attached to form a substituted orunsubstituted 5, 6, 7, or 8 membered ring.

In preferred embodiments, R^(F) and R^(G) together with the N⁺ to whichthey are attached form a five, six, seven, or eight-memberednitrogen-containing heterocyclic ring, including, but not limited to:

each optionally substituted. Preferred substituents include phenyl,CO₂R^(1T) and C(O)NR^(1V)R^(1W).

In a further aspect, R^(F) and R^(G) are independently a C₁-C₄ alkyl. Inanother embodiment, R^(F) and R^(G) are independently selected from CH₃and CH₂CH₃. In certain other aspects, R^(F) and R^(G) are the same andare substituted or unsubstituted C₁-C₄ alkyl. In yet addition aspects,R^(F) and R^(G) are the same and are methyl, ethyl, propyl, or butyl. Inyet another embodiment, R^(F) and R^(G) are the same and are CH₃ orCH₂CH₃.

In some embodiments Y⁻ is a halide anion, a carboxylate, or a sulfonate.Y⁻ can, for example, be a halide ion, a substituted or unsubstitutedalkylsulfonate, a substituted or unsubstituted arylsulfonate, asubstituted or unsubstituted alkyl or aliphatic carboxylate, asubstituted or unsubstituted aryl carboxylate, or a substituted orunsubstituted heterocyclyl carboxylate.

In certain embodiments, Y− is selected from the group consisting oftrifluoroacetate, sulfate, phosphate, acetate, fumarate, formate,carbonate, maleate, citrate, pyruvate, succinate, oxalate, a sulfonate,(for example, methanesulfonate, trifluoromethanesulfonate,toluenesulfonate such as p-toluenesulfonate, benzenesulfonate,ethanesulfonate, camphorsulfonate, 2-mesitylenesulfonate, ornaphthalenesulfonate such as 2-naphthalenesulfonate), bisulfate,malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate,adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate,L-tartrate, D-tartrate, stearate, 2-furoate, 3-furoate, napadisylate(naphthalene-1,5-disulfonate or naphthalene-1-(sulfonicacid)-5-sulfonate), edisylate (ethane-1,2-disulfonate orethane-1-(sulfonic acid)-2-sulfonate), isethionate(2-hydroxyethylsulfonate), D-mandelate, L-mandelate, propionate,tartarate, phthalate, hydrochlorate, hydrobromate, and nitrate. In oneembodiment, Y⁻ is a halide anion.

In a preferred embodiment, the anion is selected from the halide ionsbromide, chloride, or iodide.

Each preferred group stated above can be taken in combination with one,any or all other preferred groups.

In a preferred embodiment, the present invention relates to compounds ofFormula (I), or a pharmaceutically acceptable salt thereof, whereinR^(H) is an optionally substituted aryl or optionally substitutedheteroaryl selected from one of the following:

In certain preferred aspects, R^(H) is substituted or unsubstitutedphenyl.

In certain additional aspects, the compound has the Formula (II):

wherein:

Y⁻ is a pharmaceutically acceptable anion;

q is 0, 1, 2, or 3;

R^(D) is hydrogen, methyl, or ethyl; and

R^(1B) is as defined above.

In some embodiments, the compound has the Formula (II), wherein Y⁻ is ahalide anion, a carboxylate, or a sulfonate. Y⁻ can, for example, be ahalide ion, a substituted or unsubstituted alkylsulfonate, a substitutedor unsubstituted arylsulfonate, a substituted or unsubstituted alkyl oraliphatic carboxylate, a substituted or unsubstituted aryl carboxylate,or a substituted or unsubstituted heterocyclyl carboxylate.

In certain embodiments, the compound has the Formula (II), wherein Y⁻ isselected from the group consisting of trifluoroacetate, sulfate,phosphate, acetate, fumarate, formate, carbonate, maleate, citrate,pyruvate, succinate, oxalate, a sulfonate, (for example,methanesulfonate, trifluoromethanesulfonate, toluenesulfonate such asp-toluenesulfonate, benzenesulfonate, ethanesulfonate, camphorsulfonate,2-mesitylenesulfonate, or naphthalenesulfonate such as2-naphthalenesulfonate), bisulfate, malonate, xinafoate, ascorbate,oleate, nicotinate, saccharinate, adipate, formate, glycolate,L-lactate, D-lactate, aspartate, malate, L-tartrate, D-tartrate,stearate, 2-furoate, 3-furoate, napadisylate(naphthalene-1,5-disulfonate or naphthalene-1-(sulfonicacid)-5-sulfonate), edisylate (ethane-1,2-disulfonate orethane-1-(sulfonic acid)-2-sulfonate), isethionate(2-hydroxyethylsulfonate), D-mandelate, L-mandelate, propionate,tartarate, phthalate, hydrochlorate, hydrobromate, and nitrate.

In one embodiment, the compound has the Formula (II) and Y⁻ is halideanion. In a preferred embodiment, Y⁻ is selected from the halide ionsbromide, chloride, or iodide.

As will be understood, when q is 0, 1, 2, or 3, the N⁺-containing ringof Formula (II) is:

respectively. In certain preferred embodiments, q is 0. In otherpreferred aspects, q is 1. In other preferred aspects, q is 2. In yetfurther preferred aspects, q is 3.

In yet additional preferred aspects, the compound has the Formula (II)and R^(D) is hydrogen.

In further aspects, the compound has the Formula (II) and R^(D) ismethyl.

In yet other aspects, the compound has the Formula (II) and R^(D) isethyl.

In yet further aspects, the compound has the Formula (II), q is 0 andR^(D) is hydrogen.

In additional preferred aspects, the compound has the Formula (II), q is1 and R^(D) is hydrogen.

In further preferred aspects, the compound has the Formula (II) and q is2, and R^(D) is hydrogen.

In additional aspects, the compound has the Formula (II), q is 3, andR^(D) is hydrogen.

In yet additional preferred embodiments, the compound has the Formula(II), and q is 0 and R^(D) is methyl.

In additional preferred aspects, the compound has the Formula (II), q is1 and R^(D) is methyl.

In further preferred aspects, the compound has the Formula (II), q is 2and R^(D) is methyl.

In additional aspects, the compound has the Formula (II), q is 3 andR^(D) is methyl.

In further preferred embodiments, the compound has the Formula (II), qis 0 and R^(D) is ethyl.

In additional preferred aspects, the compound has the Formula (II), q is1 and R^(D) is ethyl.

In further preferred aspects, the compound has the Formula (II), q is 2and R^(D) is ethyl.

In additional aspects, the compound has the Formula (II), q is 3 andR^(D) is ethyl.

In yet an additional aspect, the compound is selected from Table Abelow, or a pharmaceutically acceptable salt thereof, wherein Y⁻ is apharmaceutically acceptable anion.

TABLE A Compound # Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

In additional preferred aspects, the compound is selected from thecompounds in the following Tables, or a pharmaceutically acceptable saltthereof, wherein Y− is as indicated or a pharmaceutically acceptableanion:

TABLE B Compound # Y⁻ Structure 1A Br⁻

1B Cl⁻

2A Br⁻

2B Cl⁻

3A Br⁻

3B Cl⁻

4A Br⁻

4B Cl⁻

5A Br⁻

5B Cl⁻

6A Br⁻

6B Cl⁻

7A Br⁻

7B Cl⁻

8A Br⁻

8B Cl⁻

9A Br⁻

9B Cl⁻

10A Br⁻

10B Cl⁻

11A Br⁻

11B Cl⁻

12A Br⁻

12B Cl⁻

13A Br⁻

13B Cl⁻

14A Br⁻

14B Cl⁻

15A Br⁻

15B Cl⁻

16A Br⁻

16B Cl⁻

17A Br⁻

17B Cl⁻

18A Br⁻

18B Cl⁻

19A Br⁻

19B Cl⁻

20A Br⁻

20B Cl⁻

21A Br⁻

21B Cl⁻

22A Br⁻

22B Cl⁻

23A Br⁻

23B Cl⁻

24A Br⁻

24B Cl⁻

25A Br⁻

25B Cl⁻

26A Br⁻

26B Cl⁻

27A Br⁻

27B Cl⁻

28 TFA⁻

29 CO₃ ⁻

30A Br⁻

30B Cl⁻

31A Br⁻

31B Cl⁻

Representative compounds according to the invention and theirenantiomers and pharmaceutically acceptable salts thereof are thoseselected from Table C below, wherein Y⁻ is a pharmaceutically acceptableanion, as defined above, and Z is either an aryl or a heteroarylstructure selected from one of the structures in Table 1, or asubstituted aryl or substituted heteroaryl structure selected from oneof the structures in Tables 2-3.

TABLE C Representative Compounds of the Invention No. Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

 10

 11

 12

 13

 14

 15

 16

 17

 18

 19

 20

 21

 22

 23

 24

 25

 26

 27

 28

 29

 30

 31

 32

 33

 34

 35

 36

 37

 38

 39

 40

 41

 42

 43

 44

 45

 46

 47

 48

 49

 50

 51

 52

 53

 54

 55

 56

 57

 58

 59

 60

 61

 62

 63

 64

 65

 66

 67

 68

 69

 70

 71

 72

 73

 74

 75

 76

 77

 78

 79

 80

 81

 82

 83

 84

 85

 86

 87

 88

 89

 90

 91

 92

 93

 94

 95

 96

 97

 98

 99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218a

218b

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

TABLE 1 Representative Z Structures No. Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

TABLE 2 Representative Z Structures No. Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

 10

 11

 12

 13

 14

 15

 16

 17

 18

 19

 20

 21

 22

 23

 24

 25

 26

 27

 28

 29

 30

 31

 32

 33

 34

 35

 36

 37

 38

 39

 40

 41

 42

 43

 44

 45

 46

 47

 48

 49

 50

 51

 52

 53

 54

 55

 56

 57

 58

 59

 60

 61

 62

 63

 64

 65

 66

 67

 68

 69

 70

 71

 72

 73

 74

 75

 76

 77

 78

 79

 80

 81

 82

 83

 84

 85

 86

 87

 88

 89

 90

 91

 92

 93

 94

 95

 96

 97

 98

 99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

TABLE 3 Representative Z Structures No. Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

Preferred compounds according to the invention and their enantiomers andpharmaceutically acceptable salts thereof are represented by Formula(III),

wherein the preferred substituent combinations R^(C), R^(D),N⁺/R^(F)/R^(G), and Z are as defined in Table 4, and Y⁻ is apharmaceutically acceptable anion as defined above. The compounds can bemade according to the methods generally described below.

TABLE 4 Preferred Combinations of R^(C), R^(D), N⁺/R^(F/)R^(G), and ZSubstituents according to Formula (III). Combination Number R^(C) R^(D)N⁺/R^(F/)R^(G) Z  1 H H

 2 Cl H

 3 CH₃ H

 4 H CH₃

 5 Cl CH₃

 6 CH₃ CH₃

 7 H CH₂CH₃

 8 Cl CH₂CH₃

 9 CH₃ CH₂CH₃

 10 H (CH₂)₂CH₃

 11 Cl (CH₂)₂CH₃

 12 CH₃ (CH₂)₂CH₃

 13 H H

 14 Cl H

 15 CH₃ H

 16 H CH₃

 17 Cl CH₃

 18 CH₃ CH₃

 19 H CH₂CH₃

 20 Cl CH₂CH₃

 21 CH₃ CH₂CH₃

 22 H (CH₂)₂CH₃

 23 Cl (CH₂)₂CH₃

 24 CH₃ (CH₂)₂CH₃

 25 H H

 26 Cl H

 27 CH₃ H

 28 H CH₃

 29 Cl CH₃

 30 CH₃ CH₃

 31 H CH₂CH₃

 32 Cl CH₂CH₃

 33 CH₃ CH₂CH₃

 34 H (CH₂)₂CH₃

 35 Cl (CH₂)₂CH₃

 36 CH₃ (CH₂)₂CH₃

 37 H H

 38 Cl H

 39 CH₃ H

 40 H CH₃

 41 Cl CH₃

 42 CH₃ CH₃

 43 H CH₂CH₃

 44 Cl CH₂CH₃

 45 CH₃ CH₂CH₃

 46 H (CH₂)₂CH₃

 47 Cl (CH₂)₂CH₃

 48 CH₃ (CH₂)₂CH₃

 49 H H

 50 Cl H

 51 CH₃ H

 52 H CH₃

 53 Cl CH₃

 54 CH₃ CH₃

 55 H CH₂CH₃

 56 Cl CH₂CH₃

 57 CH₃ CH₂CH₃

 58 H (CH₂)₂CH₃

 59 Cl (CH₂)₂CH₃

 60 CH₃ (CH₂)₂CH₃

 61 H H

 62 Cl H

 63 CH₃ H

 64 H CH₃

 65 Cl CH₃

 66 CH₃ CH₃

 67 H CH₂CH₃

 68 Cl CH₂CH₃

 69 CH₃ CH₂CH₃

 70 H (CH₂)₂CH₃

 71 Cl (CH₂)₂CH₃

 72 CH₃ (CH₂)₂CH₃

 73 H H

 74 Cl H

 75 CH₃ H

 76 H CH₃

 77 Cl CH₃

 78 CH₃ CH₃

 79 H CH₂CH₃

 80 Cl CH₂CH₃

 81 CH₃ CH₂CH₃

 82 H (CH₂)₂CH₃

 83 Cl (CH₂)₂CH₃

 84 CH₃ (CH₂)₂CH₃

 85 H H

 86 Cl H

 87 CH₃ H

 88 H CH₃

 89 Cl CH₃

 90 CH₃ CH₃

 91 H CH₂CH₃

 92 Cl CH₂CH₃

 93 CH₃ CH₂CH₃

 94 H (CH₂)₂CH₃

 95 Cl (CH₂)₂CH₃

 96 CH₃ (CH₂)₂CH₃

 97 H H

 98 Cl H

 99 CH₃ H

100 H CH₃

101 Cl CH₃

102 CH₃ CH₃

103 H CH₂CH₃

104 Cl CH₂CH₃

105 CH₃ CH₂CH₃

106 H (CH₂)₂CH₃

107 Cl (CH₂)₂CH₃

108 CH₃ (CH₂)₂CH₃

109 H H

110 Cl H

111 CH₃ H

112 H CH₃

113 Cl CH₃

114 CH₃ CH₃

115 H CH₂CH₃

116 Cl CH₂CH₃

117 CH₃ CH₂CH₃

118 H (CH₂)₂CH₃

119 Cl (CH₂)₂CH₃

120 CH₃ (CH₂)₂CH₃

121 H H

122 Cl H

123 CH₃ H

124 H CH₃

125 Cl CH₃

126 CH₃ CH₃

127 H CH₂CH₃

128 Cl CH₂CH₃

129 CH₃ CH₂CH₃

130 H (CH₂)₂CH₃

131 Cl (CH₂)₂CH₃

132 CH₃ (CH₂)₂CH₃

133 H H

134 Cl H

135 CH₃ H

136 H CH₃

137 Cl CH₃

138 CH₃ CH₃

139 H CH₂CH₃

140 Cl CH₂CH₃

141 CH₃ CH₂CH₃

142 H (CH₂)₂CH₃

143 Cl (CH₂)₂CH₃

144 CH₃ (CH₂)₂CH₃

145 H H

146 Cl H

147 CH₃ H

148 H CH₃

149 Cl CH₃

150 CH₃ CH₃

151 H CH₂CH₃

152 Cl CH₂CH₃

153 CH₃ CH₂CH₃

154 H (CH₂)₂CH₃

155 Cl (CH₂)₂CH₃

156 CH₃ (CH₂)₂CH₃

157 H H

158 Cl H

159 CH₃ H

160 H CH₃

161 Cl CH₃

162 CH₃ CH₃

163 H CH₂CH₃

164 Cl CH₂CH₃

165 CH₃ CH₂CH₃

166 H (CH₂)₂CH₃

167 Cl (CH₂)₂CH₃

168 CH₃ (CH₂)₂CH₃

169 H H

170 Cl H

171 CH₃ H

172 H CH₃

173 Cl CH₃

174 CH₃ CH₃

175 H CH₂CH₃

176 Cl CH₂CH₃

177 CH₃ CH₂CH₃

178 H (CH₂)₂CH₃

179 Cl (CH₂)₂CH₃

180 CH₃ (CH₂)₂CH₃

181 H H

182 Cl H

183 CH₃ H

184 H CH₃

185 Cl CH₃

186 CH₃ CH₃

187 H CH₂CH₃

188 Cl CH₂CH₃

189 CH₃ CH₂CH₃

190 H (CH₂)₂CH₃

191 Cl (CH₂)₂CH₃

192 CH₃ (CH₂)₂CH₃

193 H H

194 Cl H

195 CH₃ H

196 H CH₃

197 Cl CH₃

198 CH₃ CH₃

199 H CH₂CH₃

200 Cl CH₂CH₃

201 CH₃ CH₂CH₃

202 H (CH₂)₂CH₃

203 Cl (CH₂)₂CH₃

204 CH₃ (CH₂)₂CH₃

205 H H

206 Cl H

207 CH₃ H

208 H CH₃

209 Cl CH₃

210 CH₃ CH₃

211 H CH₂CH₃

212 Cl CH₂CH₃

213 CH₃ CH₂CH₃

214 H (CH₂)₂CH₃

215 Cl (CH₂)₂CH₃

216 CH₃ (CH₂)₂CH₃

217 H H

218 Cl H

219 CH₃ H

220 H CH₃

221 Cl CH₃

222 CH₃ CH₃

223 H CH₂CH₃

224 Cl CH₂CH₃

225 CH₃ CH₂CH₃

226 H (CH₂)₂CH₃

227 Cl (CH₂)₂CH₃

228 CH₃ (CH₂)₂CH₃

229 H H

230 Cl H

231 CH₃ H

232 H CH₃

233 Cl CH₃

234 CH₃ CH₃

235 H CH₂CH₃

236 Cl CH₂CH₃

237 CH₃ CH₂CH₃

238 H (CH₂)₂CH₃

239 Cl (CH₂)₂CH₃

240 CH₃ (CH₂)₂CH₃

241 H H

242 Cl H

243 CH₃ H

244 H CH₃

245 Cl CH₃

246 CH₃ CH₃

247 H CH₂CH₃

248 Cl CH₂CH₃

249 CH₃ CH₂CH₃

250 H (CH₂)₂CH₃

251 Cl (CH₂)₂CH₃

252 CH₃ (CH₂)₂CH₃

253 H H

254 Cl H

255 CH₃ H

256 H CH₃

257 Cl CH₃

258 CH₃ CH₃

259 H CH₂CH₃

260 Cl CH₂CH₃

261 CH₃ CH₂CH₃

262 H (CH₂)₂CH₃

263 Cl (CH₂)₂CH₃

264 CH₃ (CH₂)₂CH₃

265 H H

266 Cl H

267 CH₃ H

268 H CH₃

269 Cl CH₃

270 CH₃ CH₃

271 H CH₂CH₃

272 Cl CH₂CH₃

273 CH₃ CH₂CH₃

274 H (CH₂)₂CH₃

275 Cl (CH₂)₂CH₃

276 CH₃ (CH₂)₂CH₃

277 H H

278 Cl H

279 CH₃ H

280 H CH₃

281 Cl CH₃

282 CH₃ CH₃

283 H CH₂CH₃

284 Cl CH₂CH₃

285 CH₃ CH₂CH₃

286 H (CH₂)₂CH₃

287 Cl (CH₂)₂CH₃

288 CH₃ (CH₂)₂CH₃

289 H H

290 Cl H

291 CH₃ H

292 H CH₃

293 Cl CH₃

294 CH₃ CH₃

295 H CH₂CH₃

296 Cl CH₂CH₃

297 CH₃ CH₂CH₃

298 H (CH₂)₂CH₃

299 Cl (CH₂)₂CH₃

300 CH₃ (CH₂)₂CH₃

301 H H

302 Cl H

303 CH₃ H

304 H CH₃

305 Cl CH₃

306 CH₃ CH₃

307 H CH₂CH₃

308 Cl CH₂CH₃

309 CH₃ CH₂CH₃

310 H (CH₂)₂CH₃

311 Cl (CH₂)₂CH₃

312 CH₃ (CH₂)₂CH₃

313 H H

314 Cl H

315 CH₃ H

316 H CH₃

317 Cl CH₃

318 CH₃ CH₃

319 H CH₂CH₃

320 Cl CH₂CH₃

321 CH₃ CH₂CH₃

322 H (CH₂)₂CH₃

323 Cl (CH₂)₂CH₃

324 CH₃ (CH₂)₂CH₃

325 H H

326 Cl H

327 CH₃ H

328 H CH₃

329 Cl CH₃

330 CH₃ CH₃

331 H CH₂CH₃

332 Cl CH₂CH₃

333 CH₃ CH₂CH₃

334 H (CH₂)₂CH₃

335 Cl (CH₂)₂CH₃

336 CH₃ (CH₂)₂CH₃

337 H H

338 Cl H

339 CH₃ H

340 H CH₃

341 Cl CH₃

342 CH₃ CH₃

343 H CH₂CH₃

344 Cl CH₂CH₃

345 CH₃ CH₂CH₃

346 H (CH₂)₂CH₃

347 Cl (CH₂)₂CH₃

348 CH₃ (CH₂)₂CH₃

349 H H

350 Cl H

351 CH₃ H

352 H CH₃

353 Cl CH₃

354 CH₃ CH₃

355 H CH₂CH₃

356 Cl CH₂CH₃

357 CH₃ CH₂CH₃

358 H (CH₂)₂CH₃

359 Cl (CH₂)₂CH₃

360 CH₃ (CH₂)₂CH₃

361 H H

362 Cl H

363 CH₃ H

364 H CH₃

365 Cl CH₃

366 CH₃ CH₃

367 H CH₂CH₃

368 Cl CH₂CH₃

369 CH₃ CH₂CH₃

370 H (CH₂)₂CH₃

371 Cl (CH₂)₂CH₃

372 CH₃ (CH₂)₂CH₃

Each preferred embodiment described herein can be taken in combinationwith one, any or all other preferred embodiments, as though presentedherein in every permutation.

Compositions of the invention can comprise racemic mixtures, pureenantiomers, or an excess of one enantiomer over the other. For example,a composition can comprise an enantiomeric excess of at least 5, 10, 20,30, 40, 50, 60, 70, 80 or 90%. In one embodiment, the enantiomericexcess is at least 95%.

The compounds of the invention include all enantiomers which may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, as wellas their racemic and optically pure forms, and is not limited to thosedescribed herein in any of their pharmaceutically acceptable forms,including enantiomers, salts, solvates, polymorphs, solvatomorphs,hydrates, anhydrous and other crystalline forms and combinationsthereof. Likewise, all tautomeric forms are intended to be included.

Preferably, a pharmaceutical composition comprises a compound of theinvention as an R enantiomer in substantially pure form; or, apharmaceutical composition comprises a compound of the invention as an Senantiomer in substantially pure form; or, a pharmaceutical compositioncomprises a compound of the invention as enantiomeric mixtures whichcontain an excess of the R enantiomer or an excess of the S enantiomer.It is particularly preferred that the pharmaceutical compositioncontains a compound of the invention which is a substantially pureoptical isomer. For the avoidance of doubt, a compound of the inventioncan, if desired, be used in the form of solvates.

Synthesis

Compounds having Formula (I) can be prepared using methods analogous tothe following general synthetic schemes:

and,

For example,

Compounds having Formula (II) can be prepared using methods analogous tothat described in the Examples and the following synthetic schemes:

Additional Biologically Active Agents and Exogenous Large Pore ChannelAgonists

As described above, the compound or composition of the invention can beadministered with a biologically active agent. For example, one or moreadditional biologically active agents, including those typically used totreat neurogenic inflammation, may be used in combination with acompound or composition of the invention described herein. Thebiologically active agents include, but are not limited to, TRP1Areceptor agonists, TRPV1-4 receptor agonists, TRPM8 agonists, ASICagonists, P2X receptor agonists, acetaminophen, NSAIDs, glucocorticoids,narcotics, tricyclic antidepressants, amine transporter inhibitors,anticonvulsants, anti-proliferative and immune modulatory agents, anantibody or antibody fragment, an antibiotic, a polynucleotide, apolypeptide, a protein, an anti-cancer agent, a growth factor, and avaccine.

TRPV1 agonists that can be employed in the methods, kits andcompositions of the invention include, but are not limited to, any thatactivates TRPV1 receptors on nociceptors and allows for entry of atleast one inhibitor of voltage-gated ion channels (for example, acompound of the invention). A suitable TRPV1 agonist is capsaicin oranother capsaicinoids, which are members of the vanilloid family ofmolecules. Naturally occurring capsaicinoids are capsaicin itself,dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin,homocapsaicin, and nonivamide. Other suitable capsaicinoids andcapsaicinoid analogs and derivatives for use in the compositions andmethods of the present invention include naturally occurring andsynthetic capsaicin derivatives and analogs including, e.g., vanilloids(e.g., N-vanillyl-alkanedienamides, N-vanillyl-alkanedienyls, andN-vanillyl-cis-monounsaturated alkenamides), capsiate, dihydrocapsiate,nordihydrocapsiate and other capsinoids, capsiconiate,dihydrocapsiconiate and other coniferyl esters, capsiconinoid,resiniferatoxin, tinyatoxin, civamide, N-phenylmethylalkenamidecapsaicin derivatives, olvanil,N-[(4-(2-aminoethoxy)-3-methoxyphenyl)methyl]-9Z-octa-decanamide,N-oleyl-homovanillamide, triprenyl phenols (e.g., scutigeral),gingerols, piperines, shogaols, guaiacol, eugenol, zingerone, nuvanil,NE-19550, NE-21610, and NE-28345. Additional capsaicinoids, theirstructures, and methods of their manufacture are described in U.S. Pat.Nos. 7,446,226 and 7,429,673, which are hereby incorporated byreference.

Additional suitable TRPV1 agonists include but are not limited toeugenol, arvanil (N-arachidonoylvanillamine), anandamide,2-aminoethoxydiphenyl borate (2APB), AM404, resiniferatoxin, phorbol12-phenylacetate 13-acetate 20-homovanillate (PPAHV), olvanil (NE19550), OLDA (N-oleoyldopamine), N-arachidonyldopamine (NADA),6′-iodoresiniferatoxin (6′-IRTX), C18 N-acylethanolamines, lipoxygenasederivatives such as 12-hydroperoxyeicosatetraenoic acid, inhibitorcysteine knot (ICK) peptides (vanillotoxins), piperine, MSK195(N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-aminoethoxy)-3-methoxyphenyl]acetamide),JYL79(N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-(4-hydroxy-3-methoxybenzyl)thiourea),hydroxy-alpha-sanshool, 2-aminoethoxydiphenyl borate, 10-shogaol,oleylgingerol, oleylshogaol, and SU200(N-(4-tert-butylbenzyl)-N′-(4-hydroxy-3-methoxybenzyl)thiourea). Stillother TRPV1 agonists include amylocaine, articaine, benzocaine,bupivacaine, carbocaine, carticaine, chloroprocaine, cyclomethycaine,dibucaine (cinchocaine), dimethocaine (larocaine), etidocaine,hexylcaine, levobupivacaine, lidocaine, mepivacaine, meprylcaine(oracaine), metabutoxycaine, piperocaine, prilocaine, procaine(novacaine), proparacaine, propoxycaine, risocaine, ropivacaine,tetracaine (amethocaine), and trimecaine.

Suitable TRPV2-4 agonists include, but are not limited to, are 2-APB,cannabinol, diphenylboronic anhydride, insulin-like growth factor 1,lysophosphatidylcholine, lysophosphatidylinositol, probenecid,Δ9-tetrahydrocannabinol, vanillin, eugenol, cinnamaldehyde, camphor,carvacrol, thymol, citral, farnesyl diphosphate, tetrahydrocannabivarin,incensole acetate, diphenylboronic anhydride, 6-tert-butyl-m-cresol,dihydrocarveocarveol, borneol, (−)-menthol, GSK1016790A, 4α-PDH,5,6-epoxyeicosatrienoic acid, 4α-PDD, bisandrographolide, citric acid,phorbol 12-myristate 13-acetate and RN1747.

Suitable TRPM8 agonists include, but are not limited to, are menthol,icilin, eucalyptus, linalool, geraniol, hydroxy-citronellal, WS-3,WS-23, Frescolat MGA, Frescolat ML, PMD 38, CPS125, Coolact P, M8-Ag,AITC, cryosim-3 and Cooling Agent 10.

Suitable ASIC agonists include, but are not limited to,chlorophenylguanidine hydrochloride, GMQ hydrochloride,tetrahydropapaveroline (THP), reticulin, polyamine agmatine,lysophosphatidylcholine, arachidonic acid and neuropeptide SF.

Other biologically active agents which can be employed in the methods,compositions, and kits of the invention include any that activates TRP1Areceptors on nociceptors or pruriceptors and allows for entry of atleast one inhibitor of voltage-gated ion channels. Suitable TRP1Aagonists include but are not limited to cinnamaldehyde,allyl-isothiocynanate (mustard oil), diallyl disulfide, icilin, cinnamonoil, wintergreen oil, clove oil, acrolein, hydroxy-alpha-sanshool,2-aminoethoxydiphenyl borate, 4-hydroxynonenal, methylp-hydroxybenzoate, and 3′-carbamoylbiphenyl-3-yl cyclohexylcarbamate(URB597).

P2X agonists that can be employed in the methods, compositions, and kitsof the invention include any that activates P2X receptors on nociceptorsor pruriceptors and allows for entry of at least one inhibitor ofvoltage-gated ion channels. Suitable P2X agonists include but are notlimited to ATP, α,β-methylene ATP, 2-methylthio-ATP, 2′ and3′-O-(4-benzoylbenzoyl)-ATP, and ATP5′-O-(3-thiotriphosphate).

Other biologically active agents that can be used in combination withthe compounds of the invention include NSAIDs, glucocorticoids,narcotics, tricyclic antidepressants, amine transporter inhibitors,anticonvulsants, anti-proliferative and immune modulatory agents, anantibody or antibody fragment, an antibiotic, a polynucleotide, apolypeptide, a protein, an anti-cancer agent, a growth factor, and avaccine.

Non-steroidal anti-inflammatory drugs (NSAIDs) that can be administeredto a patient (e.g., a human) suffering from neurogenic inflammation incombination with a composition of the invention include, but are notlimited to, acetylsalicylic acid, amoxiprin, benorylate, benorilate,choline magnesium salicylate, diflunisal, ethenzamide, faislamine,methyl salicylate, magnesium salicylate, salicyl salicylate,salicylamide, diclofenac, aceclofenac, acemethacin, alclofenac,bromfenac, etodolac, indometacin, nabumetone, oxametacin, proglumetacin,sulindac, tolmetin, ibuprofen, alminoprofen, benoxaprofen, carprofen,dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen,flurbiprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen,naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamicacid, flufenamic acid, meclofenamic acid, tolfenamic acid,phenylbutazone, ampyrone, azapropazone, clofezone, kebuzone, metamizole,mofebutazone, oxyphenbutazone, phenazone, sulfinpyrazone, piroxicam,droxicam, lornoxicam, meloxicam, tenoxicam, and the COX-2 inhibitorscelecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, valdecoxib,and pharmaceutically acceptable salts thereof.

Glucocorticoids that can be administered to a patient (e.g., a human)suffering from neurogenic inflammation in combination with a compositionof the invention include, but are not limited to, hydrocortisone,cortisone acetate, prednisone, prednisolone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, andpharmaceutically acceptable salts thereof.

Narcotics that can be administered to a patient (e.g., a human)suffering from neurogenic inflammation in combination with a compositionof the invention include, but are not limited, to tramadol, hydrocodone,oxycodone, morphine, and pharmaceutically acceptable salts thereof.

Antiproliferative and immune modulatory agents that can be administeredto a patient (e.g., a human) suffering from neurogenic inflammation incombination with a composition of the invention include, but are notlimited to, alkylating agents, platinum agents, antimetabolites,topoisomerase inhibitors, dihydrofolate reductase inhibitors, antitumorantibiotics, antimitotic agents, aromatase inhibitors, thymidylatesynthase inhibitors, DNA antagonists, farnesyltransferase inhibitors,pump inhibitors, histone acetyltransferase inhibitors, metalloproteinaseinhibitors, ribonucleoside reductase inhibitors, TNF-alpha agonists,TNF-alpha antagonists or scavengers, interleukin 1 (IL-1) antagonists orscavengers, endothelin A receptor antagonists, retinoic acid receptoragonists, hormonal agents, antihormonal agents, photodynamic agents, andtyrosine kinase inhibitors.

The biologically active agents can be administered prior to, concurrentwith, or following administration of a composition of the invention,using any formulation, dosing, or administration known in the art thatis therapeutically effective.

Formulation of Compositions

The administration of the compounds of the invention may be by anysuitable means that results in the reduction of perceived pain sensationat the target region. The compounds of the invention may be contained inany appropriate amount in any suitable carrier substance, and aregenerally present in amounts totaling 1-99% by weight of the totalweight of the composition. The composition may be provided in a dosageform that is suitable for oral, parenteral (e.g., intravenous,intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal,sublingual, nasal, vaginal, intrathecal, epidural, or ocularadministration, or by injection, inhalation, or direct contact with thenasal or oral mucosa.

Thus, the composition may be in the form of, e.g., tablets, capsules,pills, powders, granulates, suspensions, emulsions, solutions, gelsincluding hydrogels, pastes, ointments, creams, plasters, drenches,osmotic delivery devices, suppositories, enemas, injectables, implants,sprays, or aerosols. The compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy, 22nd edition, 2013, ed. L. V. Allen,Pharmaceutical Press, Philadelphia, and Encyclopedia of PharmaceuticalTechnology, 4^(th) Edition, ed. J. Swarbrick, 2013, CRC Press, NewYork).

Each compound may be formulated in a variety of ways that are known inthe art. For example, a compound of the invention and a biologicallyactive agent as defined herein may be formulated together or separately.Desirably, a compound of the invention and a biologically active agentare formulated together for their simultaneous or near simultaneousadministration. In another embodiment, two or more biologically activeagents may be formulated together with a compound of the invention, orseparately. Other examples include, but are not limited to, two or morecompounds of the invention formulated together, wherein the compoundsare formulated together with or without one or more biologically activeagents.

The individually or separately formulated agents can be packagedtogether as a kit. Non-limiting examples include but are not limited tokits that contain, e.g., two pills, a pill and a powder, a suppositoryand a liquid in a vial, two topical creams, etc. The kit can includeoptional components that aid in the administration of the unit dose topatients, such as vials for reconstituting powder forms, syringes forinjection, customized IV delivery systems, inhalers, etc. Additionally,the unit dose kit can contain instructions for preparation andadministration of the compositions.

The kit may be manufactured as a single use unit dose for one patient,multiple uses for a particular patient (at a constant dose or in whichthe individual compounds may vary in potency as therapy progresses); orthe kit may contain multiple doses suitable for administration tomultiple patients (“bulk packaging”). The kit components may beassembled in cartons, blister packs, bottles, tubes, and the like.

Controlled Release Formulations

Each compound of the invention, alone or in combination with one or moreof the biologically active agents as described herein, can be formulatedfor controlled release (e.g., sustained or measured) administration, asdescribed in U.S. Patent Application Publication Nos. 2003/0152637 and2005/0025765, each incorporated herein by reference. For example, acompound of the invention, alone or in combination with one or more ofthe biologically active agents as described herein, can be incorporatedinto a capsule or tablet that is administered to the patient.

Any pharmaceutically acceptable vehicle or formulation suitable forlocal application and/or injection into a site to be treated (e.g., apainful surgical incision, wound, or joint), that is able to provide asustained release of compound of the invention, alone or in combinationwith one or more of the biologically active agents as described herein,may be employed to provide for prolonged elimination or alleviation ofinflammation, as needed. Controlled release formulations known in theart include specially coated pellets, polymer formulations or matricesfor surgical insertion or as sustained release microparticles, e.g.,microspheres or microcapsules, for implantation, insertion, infusion orinjection, wherein the slow release of the active medicament is broughtabout through sustained or controlled diffusion out of the matrix and/orselective breakdown of the coating of the preparation or selectivebreakdown of a polymer matrix. Other formulations or vehicles forcontrolled, sustained or immediate delivery of an agent to a preferredlocalized site in a patient include, e.g., suspensions, emulsions, gels,liposomes and any other suitable art known delivery vehicle orformulation acceptable for subcutaneous or intramuscular administration.

A wide variety of biocompatible materials may be utilized as acontrolled release carrier to provide the controlled release of acompound of the invention, alone or in combination with one or morebiologically active agents, as described herein. Any pharmaceuticallyacceptable biocompatible polymer known to those skilled in the art maybe utilized. It is preferred that the biocompatible controlled releasematerial degrade in vivo within about one year, preferably within about3 months, more preferably within about two months. More preferably, thecontrolled release material will degrade significantly within one tothree months, with at least 50% of the material degrading into non-toxicresidues, which are removed by the body, and 100% of the compound of theinvention being released within a time period within about two weeks,preferably within about 2 days to about 7 days. A degradable controlledrelease material should preferably degrade by hydrolysis, either bysurface erosion or bulk erosion, so that release is not only sustainedbut also provides desirable release rates. However, the pharmacokineticrelease profile of these formulations may be first order, zero order,bi- or multi-phasic, to provide the desired reversible localanti-nociceptive effect over the desired time period.

Suitable biocompatible polymers can be utilized as the controlledrelease material. The polymeric material may comprise biocompatible,biodegradable polymers, and in certain preferred embodiments, ispreferably a copolymer of lactic and glycolic acid. Preferred controlledrelease materials which are useful in the formulations of the inventioninclude the polyanhydrides, polyesters, co-polymers of lactic acid andglycolic acid (preferably wherein the weight ratio of lactic acid toglycolic acid is no more than 4:1 i.e., 80% or less lactic acid to 20%or more glycolic acid by weight) and polyorthoesters containing acatalyst or degradation enhancing compound, for example, containing atleast 1% by weight anhydride catalyst such as maleic anhydride. Examplesof polyesters include polylactic acid, polyglycolic acid and polylacticacid-polyglycolic acid copolymers. Other useful polymers include proteinpolymers such as collagen, gelatin, fibrin and fibrinogen andpolysaccharides such as hyaluronic acid.

The polymeric material may be prepared by any method known to thoseskilled in the art. For example, where the polymeric material iscomprised of a copolymer of lactic and glycolic acid, this copolymer maybe prepared by the procedure set forth in U.S. Pat. No. 4,293,539,incorporated herein by reference. Alternatively, copolymers of lacticand glycolic acid may be prepared by any other procedure known to thoseskilled in the art. Other useful polymers include polylactides,polyglycolides, polyanhydrides, polyorthoesters, polycaprolactones,polyphosphazenes, polyphosphoesters, polysaccharides, proteinaceouspolymers, soluble derivatives of polysaccharides, soluble derivatives ofproteinaceous polymers, polypeptides, polyesters, and polyorthoesters ormixtures or blends of any of these.

Pharmaceutically acceptable polyanhydrides which are useful in thepresent invention have a water-labile anhydride linkage. The rate ofdrug release can be controlled by the particular polyanhydride polymerutilized and its molecular weight. The polysaccharides may bepoly-1,4-glucans, e.g., starch glycogen, amylose, amylopectin, andmixtures thereof. The biodegradable hydrophilic or hydrophobic polymermay be a water-soluble derivative of a poly-1,4-glucan, includinghydrolyzed amylopectin, derivatives of hydrolyzed amylopectin such ashydroxyethyl starch (HES), hydroxyethyl amylose, dialdehyde starch, andthe like. The polyanhydride polymer may be branched or linear.

Examples of polymers which are useful in the present invention include(in addition to homopolymers and copolymers of poly(lactic acid) and/orpoly(glycolic acid)) poly[bis(p-carboxyphenoxy) propane anhydride](PCPP), poly[bis(p-carboxy)methane anhydride] (PCPM), polyanhydrides ofoligomerized unsaturated aliphatic acids, polyanhydride polymersprepared from amino acids which are modified to include an additionalcarboxylic acid, aromatic polyanhydride compositions, and co-polymers ofpolyanhydrides with other substances, such as fatty acid terminatedpolyanhydrides, e.g., polyanhydrides polymerized from monomers of dimersand/or trimers of unsaturated fatty acids or unsaturated aliphaticacids. Polyanhydrides may be prepared in accordance with the methods setforth in U.S. Pat. No. 4,757,128, incorporated herein by reference.Polyorthoester polymers may be prepared, e.g., as set forth in U.S. Pat.No. 4,070,347, incorporated herein by reference. Polyphosphoesters maybe prepared and used as set forth in U.S. Pat. Nos. 6,008,318,6,153,212, 5,952,451, 6,051,576, 6,103,255, 5,176,907 and 5,194,581,each of which is incorporated herein by reference.

Proteinaceous polymers may also be used. Proteinaceous polymers andtheir soluble derivatives include gelation biodegradable syntheticpolypeptides, elastin, alkylated collagen, alkylated elastin, and thelike. Biodegradable synthetic polypeptides includepoly-(N-hydroxyalkyl)-L-asparagine, poly-(N-hydroxyalkyl)-L-glutamine,copolymers of N-hydroxyalkyl-L-asparagine and N-hydroxyalkyl-L-glutaminewith other amino acids. Suggested amino acids include L-alanine,L-lysine, L-phenylalanine, L-valine, L-tyrosine, and the like.

In additional embodiments, the controlled release material, which ineffect acts as a carrier for a compound of the invention, alone or incombination with one or more biologically active agents as describedherein, can further include a bioadhesive polymer such as pectins(polygalacturonic acid), mucopolysaccharides (hyaluronic acid, mucin) ornon-toxic lectins or the polymer itself may be bioadhesive, e.g.,polyanhydride or polysaccharides such as chitosan.

In embodiments where the biodegradable polymer comprises a gel, one suchuseful polymer is a thermally gelling polymer, e.g., polyethylene oxide,polypropylene oxide (PEO-PPO) block copolymer such as Pluronic™ F127from BASF Wyandotte. In such cases, the local anesthetic formulation maybe injected via syringe as a free-flowing liquid, which gels rapidlyabove 30° C. (e.g., when injected into a patient). The gel system thenreleases a steady dose of a compound of the invention, alone or incombination with one or more biologically active agents as describedherein, at the site of administration.

Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystallinecellulose, starches including potato starch, calcium carbonate, sodiumchloride, lactose, calcium phosphate, calcium sulfate, or sodiumphosphate); granulating and disintegrating agents (e.g., cellulosederivatives including microcrystalline cellulose, starches includingpotato starch, croscarmellose sodium, alginates, or alginic acid);binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, taste masking agents (such as hydroxypropyl methylcellulose,hydroxypropyl cellulose), and the like.

One or more compounds of the invention and one or more biologicallyactive agents, as defined herein, may be mixed together in a tablet,capsule, or other vehicle, or may be partitioned. In one example, acompound of the invention is contained on the inside of the tablet, andthe biologically active agent is on the outside of the tablet, such thata substantial portion of the biologically active agent is released priorto the release of the compound of the invention.

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders, granulates, and pellets may be prepared using the ingredientsmentioned above under tablets and capsules in a conventional mannerusing, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Formulations for oral administration to the mouth may also be providedas a mouthwash, an oral spray, oral rinse solution, oral ointment, ororal gel.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of compounds, or by incorporating the compound into anappropriate matrix. A controlled release coating may include one or moreof the coating substances mentioned above and/or, e.g., shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Generally, when administered to a human, the oral dosage of any of thecompounds of the combination of the invention will depend on the natureof the compound, and can readily be determined by one skilled in theart. Typically, such dosage is normally about 0.001 mg to 2000 mg perday, desirably about 1 mg to 1000 mg per day, and more desirably about 5mg to 500 mg per day. Dosages up to 200 mg per day may be necessary.

Administration of each drug in a combination therapy, as describedherein, can, independently, be one to four times daily for one day toone year, and may even be for the life of the patient. Chronic,long-term administration will be indicated in many cases.

Parenteral Formulations

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the compound isdissolved, suspended, or otherwise provided (e.g., in a liposome orother microparticulate). Such liquids may additional contain otherpharmaceutically acceptable ingredients, such as anti-oxidants, buffers,preservatives, stabilizers, bacteriostats, suspending agents, thickeningagents, and solutes which render the formulation isotonic with the blood(or other relevant bodily fluid) of the intended recipient. Examples ofexcipients include, for example, water, alcohols, polyols, glycerol,vegetable oils, and the like. Examples of suitable isotonic carriers foruse in such formulations include Sodium Chloride Injection, Ringer'sSolution, or Lactated Ringer's Injection. Typically, the concentrationof the compound in the liquid is from about 1 ng/ml to about 10 μg/ml,for example from about 10 ng/ml to about 1 μg/ml. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

Topical Formulations

The compositions of the invention, alone or in combination with one ormore of the biologically active agents described herein, can also beadapted for topical use with a topical vehicle containing from between0.0001% and 25% (w/w) or more of active ingredient(s).

In a preferred combination, the active ingredients are preferably eachfrom between 0.0001% to 10% (w/w), more preferably from between 0.0005%to 4% (w/w) active agent. The topical formulation, including but notlimited to a cream, gel, or ointment, can be applied one to four timesdaily, or as needed. Performing the methods described herein, thetopical vehicle containing the composition of the invention, or acombination therapy containing a composition of the invention ispreferably applied to the site of inflammation on the patient. Forexample, a cream may be applied to the hands of a patient suffering fromarthritic fingers.

The compositions can be formulated using any dermatologically acceptablecarrier. Exemplary carriers include a solid carrier, such as alumina,clay, microcrystalline cellulose, silica, or talc; and/or a liquidcarrier, such as an alcohol, a glycol, or a water-alcohol/glycol blend.The therapeutic agents may also be administered in liposomalformulations that allow therapeutic agents to enter the skin. Suchliposomal formulations are described in U.S. Pat. Nos. 5,169,637;5,000,958; 5,049,388; 4,975,282; 5,194,266; 5,023,087; 5,688,525;5,874,104; 5,409,704; 5,552,155; 5,356,633; 5,032,582; 4,994,213;8,822,537, and PCT Publication No.

WO 96/40061. Examples of other appropriate vehicles are described inU.S. Pat. Nos. 4,877,805, 8,822,537, and EP Publication No. 0586106A1.Suitable vehicles of the invention may also include mineral oil,petrolatum, polydecene, stearic acid, isopropyl myristate, polyoxyl 40stearate, stearyl alcohol, or vegetable oil.

The composition can further include a skin penetrating enhancer, such asthose described in “Percutaneous Penetration enhancers”, (eds. Smith E Wand Maibach H I. CRC Press 1995). Exemplary skin penetrating enhancersinclude alkyl (N,N-disubstituted amino alkanoate) esters, such asdodecyl 2-(N,N dimethylamino) propionate (DDAIP), which is described inpatents U.S. Pat. Nos. 6,083,996 and 6,118,020, which are bothincorporated herein by reference; a water-dispersible acid polymer, suchas a polyacrylic acid polymer, a carbomer (e.g., Carbopol™ or Carbopol940P™, available from B. F. Goodrich Company (Akron, Ohio)), copolymersof polyacrylic acid (e.g., Pemulen™ from B. F. Goodrich Company orPolycarbophil™ from A. H. Robbins, Richmond, Va.; a polysaccharide gum,such as agar gum, alginate, carrageenan gum, ghatti gum, karaya gum,kadaya gum, rhamsan gum, xanthan gum, and galactomannan gum (e.g., guargum, carob gum, and locust bean gum), as well as other gums known in theart (see for instance, Industrial Gums: Polysaccharides & TheirDerivatives, Whistler R. L., BeMiller J. N. (eds.), 3rd Ed. AcademicPress (1992) and Davidson, R. L., Handbook of Water-Soluble Gums &Resins, McGraw-Hill, Inc., N.Y. (1980)); or combinations thereof.

Other suitable polymeric skin penetrating enhancers are cellulosederivatives, such as ethyl cellulose, methyl cellulose, hydroxypropylcellulose. Additionally, known transdermal penetrating enhancers canalso be added, if desired. Illustrative are dimethyl sulfoxide (DMSO)and dimethyl acetamide (DMA), 2-pyrrolidone, N,N-diethyl-m-toluamide(DEET), 1-dodecylazacycloheptane-2-one (Azone™, a registered trademarkof Nelson Research), N,N-dimethylformamide, N-methyl-2-pyrrolidone,calcium thioglycolate and other enhancers such as dioxolanes, cyclicketones, and their derivatives and so on.

Also illustrative are a group of biodegradable absorption enhancerswhich are alkyl N,N-2-(disubstituted amino) alkanoates as described inU.S. Pat. Nos. 4,980,378 and 5,082,866, which are both incorporatedherein by reference, including: tetradecyl (N,N-dimethylamino) acetate,dodecyl (N,N-dimethylamino) acetate, decyl (N,N-dimethylamino) acetate,octyl (N,N-dimethylamino) acetate, and dodecyl (N,N-diethylamino)acetate.

Particularly preferred skin penetrating enhancers include isopropylmyristate; isopropyl palmitate; dimethyl sulfoxide; decyl methylsulfoxide; dimethylalanine amide of a medium chain fatty acid; dodecyl2-(N,N-dimethylamino) propionate or salts thereof, such as its organic(e.g., hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acidaddition salts) and inorganic salts (e.g., acetic, benzoic, salicylic,glycolic, succinic, nicotinic, tartaric, maleic, malic, pamoic,methanesulfonic, cyclohexanesulfamic, picric, and lactic acid additionsalts), as described in U.S. Pat. No. 6,118,020; and alkyl2-(N,N-disubstituted amino)-alkanoates, as described in U.S. Pat. Nos.4,980,378 and 5,082,866.

The skin penetrating enhancer in this composition by weight would be inthe range of 0.5% to 10% (w/w). The most preferred range would bebetween 1.0% and 5% (w/w). In another embodiment, the skin penetratingenhancer comprises between 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, or 4%-5%, (w/w)of the composition.

The compositions can be provided in any useful form. For example, thecompositions of the invention may be formulated as solutions, emulsions(including microemulsions), suspensions, creams, ointments, foams,lotions, gels, powders, or other typical solid, semi-solid, or liquidcompositions (e.g., topical sprays) used for application to the skin orother tissues where the compositions may be used. Such compositions maycontain other ingredients typically used in such products, such ascolorants, fragrances, thickeners (e.g., xanthan gum, a fatty acid, afatty acid salt or ester, a fatty alcohol, a modified cellulose, amodified mineral material, Krisgel 100™, or a synthetic polymer),antimicrobials, solvents, surfactants, detergents, gelling agents,antioxidants, fillers, dyestuffs, viscosity-controlling agents,preservatives, humectants, emollients (e.g., natural or synthetic oils,hydrocarbon oils, waxes, or silicones), hydration agents, chelatingagents, demulcents, solubilizing excipients, adjuvants, dispersants,skin penetrating enhancers, plasticizing agents, preservatives,stabilizers, demulsifiers, wetting agents, sunscreens, emulsifiers,moisturizers, astringents, deodorants, and optionally includinganesthetics, anti-itch actives, botanical extracts, conditioning agents,darkening or lightening agents, glitter, humectants, mica, minerals,polyphenols, silicones or derivatives thereof, sunblocks, vitamins, andphytomedicinals.

The compositions can also include other like ingredients to provideadditional benefits and improve the feel and/or appearance of thetopical formulation. Specific classes of additives commonly use in theseformulations include: isopropyl myristate, sorbic acid NF powder,polyethylene glycol, phosphatidylcholine (including mixtures ofphosphatidylcholine, such as phospholipon G), Krisgel 100™ distilledwater, sodium hydroxide, decyl methyl sulfoxide (as a skin penetratingenhancer), menthol crystals, lavender oil, butylated hydroxytoluene,ethyl diglycol reagent, and 95% percent (190 proof) ethanol.

Formulations for Ophthalmic Administration

The compounds of the invention can also be formulated with anophthalmically acceptable carrier in sufficient concentration so as todeliver an effective amount of the active compound or compounds to theoptic nerve site of the eye. Preferably, the ophthalmic, therapeuticsolutions contain one or more of the active compounds in a concentrationrange of approximately 0.0001% to approximately 5% (weight by volume)and more preferably approximately 0.0005% to approximately 0.1% (weightby volume).

An ophthalmically acceptable carrier does not cause significantirritation to the eye and does not abrogate the pharmacological activityand properties of the charged sodium channel blockers.

Ophthalmically acceptable carriers are generally sterile, essentiallyfree of foreign particles, and generally have a pH in the range of 5-8.Preferably, the pH is as close to the pH of tear fluid (7.4) aspossible. Ophthalmically acceptable carriers are, for example, sterileisotonic solutions such as isotonic sodium chloride or boric acidsolutions. Such carriers are typically aqueous solutions contain sodiumchloride or boric acid. Also useful are phosphate buffered saline (PBS)solutions.

Various preservatives may be used in the ophthalmic preparation.Preferred preservatives include, but are not limited to, benzalkoniumpotassium, chlorobutanol, thimerosal, phenylmercuric acetate, andphenylmercuric nitrate. Likewise, various preferred vehicles may be usedin such ophthalmic preparation. These vehicles include, but are notlimited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose,poloxamers, carboxymethyl cellulose and hydroxyethyl cellulose.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, etc., mannitol and glycerin, or any other suitableophthalmically acceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. Accordingly, buffersinclude but are not limited to, acetate buffers, citrate buffers,phosphate buffers, and borate buffers. Acids or bases may be used toadjust the pH of these formulations as needed. Ophthalmically acceptableantioxidants can also be include. Antioxidants include but are notlimited to sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole, and butylated hydroxytoluene.

Formulations for Nasal and Inhalation Administration

The pharmaceutical compositions of the invention can be formulated fornasal or intranasal administration. Formulations suitable for nasaladministration, when the carrier is a solid, include a coarse powderhaving a particle size, for example, in the range of approximately 20 to500 microns which is administered by rapid inhalation through the nasalpassage. When the carrier is a liquid, for example, a nasal spray or asnasal drops, one or more of the formulations can be admixed in anaqueous or oily solution and inhaled or sprayed into the nasal passage.

For administration by inhalation, the active ingredient can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount, Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of, forexample, gelatin or blisters of, for example, laminated aluminum foil,for use in an inhaler or insufflator. Powder blend formulationsgenerally contain a powder mix for inhalation of the compound of theinvention and a suitable powder base (carrier/diluent/excipientsubstance) such as mono-, di or ploy-saccharides (e.g. lactose orstarch). Use of lactose is preferred. In one embodiment, each capsule orcartridge may contain between about 2 ug to about 100 mg of the compoundof formula (I) optionally in combination with another therapeuticallyactive ingredient. In a preferred embodiment, each capsule or cartridgemay contain between about 10 ug to about 50 mg of the compound offormula (I) optionally in combination with another therapeuticallyactive ingredient. In another embodiment, each capsule or cartridge maycontain between about 20 ug to about 10 mg of the compound of formula(I) optionally in combination with another therapeutically activeingredient. Alternatively, the compound of the invention may bedelivered without excipients.

Suitably, the packaging/medicament dispenser is of a type selected fromthe group consisting of a reservoir dry powder inhaler (RDPI), singleuse inhaler (capsule or blister inhaler), a multi-dose dry powderinhaler (MDPI), and a metered dose inhaler (MDI).

Solutions or suspensions for use in a pressurized container, pump,spray, atomizer, or nebulizer can be formulated to contain an aqueousmedium, ethanol, aqueous ethanol, or a suitable alternative agent fordispersing, solubilizing, or extending release of the activeingredient(s); a propellant as solvent; and/or a surfactant, such assorbitan trioleate, oleic acid, or an oligolactic acid.

Compositions formulated for nasal or inhalation administration mayinclude one or more taste-masking agents such as flavoring agents,sweeteners, and other strategies, such as sucrose, dextrose, andlactose, carboxylic acids, menthol, amino acids or amino acidderivatives such as arginine, lysine, and monosodium glutamate, and/orsynthetic flavor oils and flavoring aromatics and/or natural oils,extracts from plants, leaves, flowers, fruits, etc. and combinationsthereof. These may include cinnamon oils, oil of wintergreen, peppermintoils, clover oil, bay oil, anise oil, eucalyptus, vanilla, citrus oilsuch as lemon oil, orange oil, grape and grapefruit oil, fruit essencesincluding apple, peach, pear, strawberry, raspberry, cherry, plum,pineapple, apricot, etc. Additional sweeteners include sucrose,dextrose, aspartame, acesulfame-K, sucralose and saccharin, organicacids (by non-limiting example citric acid and aspartic acid). Suchflavors may be present at from about 0.05 to about 4 percent by weight,and may be present at lower or higher amounts as a factor of one or moreof potency of the effect on flavor, solubility of the flavorant, effectsof the flavorant on solubility or other physicochemical orpharmacokinetic properties of other formulation components, or otherfactors.

Indications

The compounds, compositions, methods, and kits of the invention can beused to treat pain, cough or itch associated with any of a number ofconditions, including trigeminal trophic syndrome, erythromelalgia, backand neck pain, lower back pain, cancer pain, gynecological and laborpain, abdominal wall pain, chronic abdominal wall pain, fibromyalgia,allergic rhinitis, arthritis, rheumatoid arthritis, osteoarthritis,rheumatological pains, orthopedic pains, acute and post herpeticneuralgia and other neuropathic pains (including peripheral neuropathy),sickle cell crises, muscle pain, vulvodynia, rectal pain, Levator anisyndrome, proctalgia fugax, peri-anal pain, hemorrhoid pain, stomachpain, ulcers, inflammatory bowel disease, irritable bowel disease,irritable bowel syndrome, oral mucositis, esophagitis, interstitialcystitis, urethritis and other urological pains, dental pain, burn pain,headaches, ophthalmic irritation, conjunctivitis (e.g., allergicconjunctivitis), eye redness, dry eye, dry eye syndrome (chronic ocularpain), complex regional pain syndrome, acute postoperative pain,postoperative pain, post-surgical ocular pain, and procedural pain(i.e., pain associated with injections, draining an abscess, surgery,dental procedures, ophthalmic procedures, ophthalmic irritation,conjunctivitis (e.g., allergic conjunctivitis), eye redness, dry eye,arthroscopies and use of other medical instrumentation, cosmeticsurgical procedures, dermatological procedures, setting fractures,biopsies, and the like).

Since a subclass of nociceptors mediate itch sensation, the compounds,compositions, methods, and kits of the invention can also be used totreat itch in patients with conditions like pruritus (including, but notlimited to, brachioradial, chronic idiopathic, genital/anal, notalgiaparesthetica, and scalp), allergic dermatitis, atopic dermatitis,contact dermatitis, poison ivy, infections, parasites, insect bites,pregnancy, metabolic disorders, liver or renal failure, drug reactions,allergic reactions, eczema, hand eczema, genital and anal itch,hemorrhoid itch, and cancer.

Since a subclass of nociceptors can initiate aberrant cough reflexes,the compounds, compositions, methods, and kits of the invention can alsobe used to treat cough in patients with conditions like asthma, COPD,asthma-COPD overlap syndrome (ACOS), interstitial pulmonary fibrosis(IPF), idiopathic pulmonary fibrosis, post viral cough, post-infectioncough, chronic idiopathic cough and lung cancer.

The compounds, compositions, methods, and kits of the invention can alsobe used to treat neurogenic inflammation and neurogenic inflammatorydisorders. Inflammation is a complex set of responses to harmful stimulithat results in localized redness, swelling, and pain. Inflammation canbe innate or adaptive, the latter driven by antigens and is mediated byimmune cells (immune-mediated inflammation). Neurogenic inflammationresults from the efferent functions of pain-sensing neurons(nociceptors), wherein neuropeptides and other chemicals that arepro-inflammatory mediators are released from the peripheral terminals ofthe nociceptors when they are activated. This release process ismediated by calcium influx and exocytosis of peptide containingvesicles, and the pro-inflammatory neuropeptides include substance P,neurokinin A and B (collectively known as tachykinins), calcitoningene-related peptide (CGRP), and vasoactive intestinal polypeptide(VIP).

The release of peripheral terminal chemicals stimulate a variety ofinflammatory responses. First, the release of substance P can result inan increase in capillary permeability such that plasma proteins leakfrom the intravascular compartment into the extracellular space (plasmaextravasation), causing edema. This can be detected as a wheal (a firm,elevated swelling of the skin) which is one component of a triad ofinflammatory responses—wheal, red spot, and flare—known as the Lewistriple response. Second, the release of CGRP causes vasodilation,leading to increased blood flow. This can be detected as a flare, whichis another component of the Lewis triple response.

Substance P also has a pro-inflammatory action on immune cells (e.g.macrophages, T-cells, mast cells, and dendritic cells) via theirneurokinin-1 (NK1) receptor. This effect has been documented in allergicrhinitis, gastritis, and colitis, and represents an interface betweenthe neurogenic and immune-mediated components of inflammation. SubstanceP released from one nociceptor may also act on NK1 receptors onneighboring nociceptors to sensitize or activate them, causing a spreadof activation and afferent/efferent function. These efferent functionsof nociceptors can be triggered by: 1) Direct activation of a nociceptorterminal by a peripheral adequate stimulus applied to the terminal (e.g.a pinch); 2) Indirect antidromic activation of a non-stimulatednociceptor terminal by the axon reflex, wherein action potential inputfrom one terminal of a nociceptor, upon reaching a converging axonalbranch point in the periphery, results in an action potential travelingfrom the branch point down to the peripheral terminal of anon-stimulated terminal; and 3) Activation as a result of activity innociceptor central terminals in the CNS traveling to the periphery(e.g., primary afferent depolarization of central terminals produced byGABA can be sufficient to initiate action potentials traveling the“wrong way”).

Genomic analysis of lung resident ILC2 cells has revealed expression ofreceptors for several neuropeptides released by sensory neurons,including SP, CGRP and VIP, providing an opportunity for nociceptors todirectly communicate with these cells. In particular, VIP is found to beexpressed in NaV1.8+ nodose ganglion neurons, including lung afferentsin OVA-exposed mice. Cultured nodose ganglion neurons stimulated withcapsaicin or IL5 also released VIP while BALF from OVA-exposed micecontained elevated VIP compared to vehicle-challenged mice (Talbot etal., Neuron. 2015 Jul. 15; 87(2): 341-354). These data indicate that VIPis released in the inflamed lung and can be blocked by silencing neuronswith charged sodium channel blockers of the present invention. Inaddition, when CD4+ T cells cultured under T_(H)2 skewing conditionswere exposed to recombinant mouse VIP, the transcript levels of IL-13and IL-5 increased, suggesting that VIP contributes to the competence ofT_(H)2 cells to transcribe these type II regulatory cytokines.

Immune mediator release from immune cells can also activate nociceptors.Mast cells are found close to primary nociceptive neurons and contributeto nociceptor sensitization in a number of contexts. Injection of thesecretagogue compound 48/80 promotes degranulation of mast cells in thedura and leads to excitation of meningeal nociceptors. Mast celldegranulation also contributes to the rapid onset of nerve growthfactor-induced thermal hyperalgesia.

Macrophages contribute to nociceptor sensitization by releasing severalsoluble mediators. Expression of the chemokine macrophage inflammatoryprotein-la (MIP-la) and its receptors CCR1 and CCR5 is increased inmacrophages and Schwann cells after partial ligation of the sciaticnerve and contributes to the development of neuropathic pain.Lymphocytes contribute to the sensitization of peripheral nociceptors. Tcells infiltrate the sciatic nerve and dorsal root ganglion (DRG) afternerve injury. Hyperalgesia and allodynia induced by nerve injury aremarkedly attenuated or abrogated in rodents lacking T cells and theimmunosuppressant rapamycin attenuates neuropathic pain in rats, partlyowing to an effect on T cells. Among the subsets of T cells, type 1 and2 helper T cells (T_(H)1 and T_(H)2 cells) have been shown to havedifferent roles in neuropathic pain. T_(H)1 cells facilitate neuropathicpain behavior by releasing proinflammatory cytokines (IL-2 andinterferon-γ (IFNγ)), whereas T_(H)2 cells inhibit it by releasinganti-inflammatory cytokines (IL-4, IL-10 and IL-13). The complementsystem also has a role in inflammatory hyperalgesia and neuropathicpain. C5a, an anaphylatoxin, is an important effector of the complementcascade and upon binding to C5aR1 receptors on neutrophils it becomes apotent neutrophil attractant (Ren & Dubner, Nat. Med. 16:1267-1276(2010)).

Bacterial infections have been shown to directly activate nociceptors,and that the immune response mediated through TLR2, MyD88, T cells, Bcells, and neutrophils and monocytes is not necessary for Staphylococcusaureus-induced pain in mice (Chiu et al., Nature 501:52-57 (2013)).Mechanical and thermal hyperalgesia in mice is correlated with livebacterial load rather than tissue swelling or immune activation.Bacteria induce calcium flux and action potentials in nociceptorneurons, in part via bacterial N-formylated peptides and thepore-forming toxin α-haemolysin, through distinct mechanisms. Specificablation of Nav1.8-lineage neurons, which include nociceptors, abrogatedpain during bacterial infection, but concurrently increased local immuneinfiltration and lymphadenopathy of the draining lymph node. Thus,bacterial pathogens produce pain by directly activating sensory neuronsthat modulate inflammation, an unsuspected role for the nervous systemin host-pathogen interactions. Data from Talbot et al., (Neuron. 2015Jul. 15; 87(2): 341-354.) have also suggested that nociceptors areactivated during exposure to allergens in sensitized animals.

In certain disorders, neurogenic inflammation contributes to theperipheral inflammation elicited by tissue injury, autoimmune disease,infection, and exposure to irritants in soft tissue, skin, therespiratory system, joints, the urogenital and GI tract, the liver, andthe brain. Neurogenic inflammatory disorders include, but are notlimited to, allergic inflammation, inflammatory bowel disease,interstitial cystitis, atopic dermatitis, asthma, conjunctivitis,arthritis, colitis, contact dermatitis, diabetes, eczema, cystitis,gastritis, migraine headache, psoriasis, rhinitis, rosacea, sunburn,pancreatitis, chronic cough, chronic rhinosinusistis, traumatic braininjury, polymicrobial sepsis, tendinopathies, chronic urticaria,rheumatic disease, acute lung injury, exposure to irritants, inhalationof irritants, pollutants, or chemical warfare agents, as describedherein.

Assessment of Pain, Cough, Itch, and Neurogenic Inflammation

In order to measure the efficacy of any of the compounds, compositions,methods, and kits of the invention in the treatment of pain associatedwith musculoskeletal, immunoinflammatory and neuropathic disorders, ameasurement index may be used. Indices that are useful include a visualanalog scale (VAS), a Likert scale, categorical pain scales,descriptors, the Lequesne index, the WOMAC index, and the AUSCAN index,each of which is well known in the art. Such indices may be used tomeasure pain, itch, function, stiffness, or other variables.

A visual analog scale (VAS) provides a measure of a one-dimensionalquantity. A VAS generally utilizes a representation of distance, such asa picture of a line with hash marks drawn at regular distance intervals,e.g., ten 1-cm intervals. For example, a patient can be asked to rank asensation of pain or itch by choosing the spot on the line that bestcorresponds to the sensation of pain or itch, where one end of the linecorresponds to “no pain” (score of 0 cm) or “no itch” and the other endof the line corresponds to “unbearable pain” or “unbearable itch” (scoreof 10 cm). This procedure provides a simple and rapid approach toobtaining quantitative information about how the patient is experiencingpain or itch. VAS scales and their use are described, e.g., in U.S. Pat.Nos. 6,709,406 and 6,432,937.

A Likert scale similarly provides a measure of a one-dimensionalquantity. Generally, a Likert scale has discrete integer values rangingfrom a low value (e.g., 0, meaning no pain) to a high value (e.g., 7,meaning extreme pain). A patient experiencing pain is asked to choose anumber between the low value and the high value to represent the degreeof pain experienced. Likert scales and their use are described, e.g., inU.S. Pat. Nos. 6,623,040 and 6,766,319.

The Lequesne index and the Western Ontario and McMaster Universities(WOMAC) osteoarthritis index assess pain, function, and stiffness in theknee and hip of OA patients using self-administered questionnaires. Bothknee and hip are encompassed by the WOMAC, whereas there is one Lequesnequestionnaire for the knee and a separate one for the hip. Thesequestionnaires are useful because they contain more information contentin comparison with VAS or Likert. Both the WOMAC index and the Lequesneindex questionnaires have been extensively validated in OA, including insurgical settings (e.g., knee and hip arthroplasty). Their metriccharacteristics do not differ significantly.

The AUSCAN (Australian-Canadian hand arthritis) index employs a valid,reliable, and responsive patient self-reported questionnaire. In oneinstance, this questionnaire contains 15 questions within threedimensions (Pain, 5 questions; Stiffness, 1 question; and Physicalfunction, 9 questions). An AUSCAN index may utilize, e.g., a Likert or aVAS scale.

Indices that are useful in the methods, compositions, and kits of theinvention for the measurement of pain include the Pain Descriptor Scale(PDS), the Visual Analog Scale (VAS), the Verbal Descriptor Scales(VDS), the Numeric Pain Intensity Scale (NPIS), the Neuropathic PainScale (NPS), the Neuropathic Pain Symptom Inventory (NPSI), the PresentPain Inventory (PPI), the Geriatric Pain Measure (GPM), the McGill PainQuestionnaire (MPQ), mean pain intensity (Descriptor DifferentialScale), numeric pain scale (NPS) global evaluation score (GES) theShort-Form McGill Pain Questionnaire, the Minnesota MultiphasicPersonality Inventory, the Pain Profile and Multidimensional PainInventory, the Child Heath Questionnaire, and the Child AssessmentQuestionnaire.

Itch can be measured by subjective measures (VAS, Lickert, descriptors).Another approach is to measure scratch which is an objective correlateof itch using a vibration transducer or movement-sensitive meters.

Cough can be measured by standard questionnaires like the LeicesterCough Questionnaire as well as validated objective instruments tomeasure cough frequency (e.g. VitaloJAK).

EXAMPLES

The following examples are intended to illustrate the invention and arenot intended to limit it.

General Abbreviation Definitions

ACN acetonitrile

AcOH acetic acid

aq. aqueous

Bn benzyl

Boc tert-butyloxycarbonyl

brine saturated sodium chloride solution in water

° C. degrees Celsius

chemical shift (ppm)

d deuterium

DCM dichloromethane

DIPEA diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMSO dimethyl sulfoxide

ESI electrospray ionization

Et₂O diethyl ether

EtOAc ethyl acetate

g gram

h hour

HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide

MeOH methanol

mHz megahertz

min minute

ml milliliter

mmol millimole

MS mass spectrometry

m/z mass to charge ratio

NMR nuclear magnetic resonance

Pet ether petroleum ether

RT room temperature

TEA triethylamine

TLC thin layer chromatography

UV ultraviolet light

1. Synthesis of N-benzyl-2-((2,6-dimethylphenyl) amino)-N,N-diethyl-2-oxoethan-1-aminium bromide

To a stirred solution of 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide (0.15 g, 0.640 mmol, 1 eq; Comb-Blocks, Inc., San Diego,Calif. Catalogue no: QA-3221) in ACN (3 mL) was added benzyl bromide(0.164 g, 0.960 mmol, 1.5 eq) and the reaction mixture was stirred for12 h at 75° C. as progress of the reaction was monitored by TLC (mobilephase 10% Methanol in DCM; visualization by UV). The reaction mixturewas evaporated under reduced pressure to afford crude product, which wastriturated with EtOAc (2×5 mL) to afford productN-benzyl-2-((2,6-dimethylphenyl)amino)-N,N-diethyl-2-oxoethan-1-aminiumbromide (0.08 g) as a white solid. MS (ESI): m/z 325.2 [M]⁺. ¹H NMR (400MHz, DMSO-d₆) δ 10.03 (br s, 1H), 7.66-7.39 (m, 5H), 7.13 (s, 3H), 4.82(s, 2H), 4.17 (s, 2H), 3.52 (q, J=6.7 Hz, 4H), 2.20 (s, 6H), 1.42 (br t,J=6.9 Hz, 6H).

2. Synthesis of N-benzyl-2-((2,6-dimethylphenyl) amino)-N,N-dimethyl-2-oxoethan-1-amminium bromide

Synthesis of Intermediate 2-bromo-N-(2,6-dimethylphenyl)acetamide

To a suspension of 2,6-dimethylaniline (30.5 mL, 247.56 mmol, 1.0 eq) inwater (300 mL) was added bromoacetyl bromide (23.8 mL, 272.31 mmol, 1.1eq) at 10° C. The reaction mixture was maintained at pH 9-10 with 15%Na₂CO₃ (aq.) solution for 1 hour as the progress of the reaction wasmonitored by TLC (mobile phase: 30% EtOAc in hexane, visualization byUV). The reaction mixture was extracted with EtOAc (2×600 mL) and thecombined organic extracts were washed with brine (200 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The resultingcrude product was triturated with Et₂O (2×200 mL) to afford2-bromo-N-(2,6-dimethylphenyl) acetamide (21 g) as a white solid. MS(ESI): m/z 244.02 [M+2]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.75 (br s, 1H),7.20-7.02 (m, 3H), 4.07 (s, 2H), 2.24 (s, 6H).

Synthesis of Intermediate 2-(dimethylamino)-N-(2,6-dimethylphenyl)acetamide

To a stirred solution of 2-bromo-N-(2,6-dimethylphenyl) acetamide (5 g,20.7 mmol, 1.0 eq) in EtOAc (75 mL) was added a 2.0 M solution ofdimethyl amine in THE (51.8 mL, 103.5 mmol, 5 eq) and the reactionmixture was stirred at 75° C. for 18 h in in steel bomb. The reactionmixture was quenched with water (100 mL) and extracted with EtOAc (2×200mL). The combined organic extracts were dried over Na₂SO₄, filtered andconcentrated under reduced pressure to afford2-(dimethylamino)-N-(2,6-dimethylphenyl) acetamide (4.1 g). ¹H NMR (400MHz, CDCl₃-d) δ ppm 8.63 (1H, br s), 7.02-7.15 (3H, m), 3.15 (2H, s),2.45 (6H, s), 2.24 (6H, s).

Synthesis of N-benzyl-2-((2,6-dimethylphenyl) amino)-N,N-dimethyl-2-oxoethan-1-aminium bromide

To a stirred solution of 2-(dimethylamino)-N-(2,6-dimethylphenyl)acetamide (0.2 g, 0.969 mmol, 1.0 eq) in ACN (3 mL) was added benzylbromide (0.23 mL, 1.936 mmol, 2.0 eq) and the reaction mixture wasstirred at 90° C. for 16 h as progress of the reaction was monitored byTLC (mobile phase: 10% MeOH in DCM, visualization by UV). The reactionmixture was cooled to room temperature and concentrated under reducedpressure to afford crude product, which was triturated with EtOAc (5 mL)to afford productN-benzyl-2-((2,6-dimethylphenyl)amino)-N,N-dimethyl-2-oxoethan-1-aminiumbromide (0.14 g). MS (ESI): m/z 297.2 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δppm 9.95 (1H, s), 7.49-7.66 (5H, m), 7.06-7.19 (3H, m), 4.81 (2H, s),4.30 (2H, s), 3.26 (6H, s), 2.19 (6H, s).

3. Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)azepan-1-ium bromide

Synthesis of Intermediate 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)acetamide

Synthesis of intermediate 2-bromo-N-(2,6-dimethylphenyl) acetamide wasas described above for Compound 2. To a stirred solution of2-bromo-N-(2,6-dimethylphenyl)acetamide (0.5 g, 1.9 mmol, 1.0 eq) in ACN(5.0 mL) was added K₂CO₃ (0.639 g, 4.6 mmol, 2.5 eq) and azepane (0.4mL, 4.0 mmol, 2.0 eq), and the mixture was stirred for 20 h at 90° C. asprogress of the reaction was monitored by TLC (mobile phase: 5% MeOH inDCM, visualization by UV). The crude reaction was concentrated underreduced pressure and the resulting residue was partitioned between waterand EtOAc (2×50 mL). The combined organic extracts were washed withbrine solution, dried over Na₂SO₄, filtered and concentrated underreduced pressure to afford crude product, which was triturated withEtOAc (20 mL) to afford 2-(azepan-1-yl)-N-(2,6-dimethylphenyl) acetamide(0.47 g). MS (ESI): m/z 261.57 [M+H]⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ8.83 (br s, 1H), 7.09 (s, 3H), 3.32 (s, 2H), 2.93-2.73 (m, 4H), 2.24 (s,6H), 1.87-1.52 (m, 9H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)azepan-1-ium bromide

To a stirred solution of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)acetamide (0.2 g, 0.8 mmol, 1.0 eq) in ACN (1.0 mL) was added benzylbromide (0.1 mL, 0.6 mmol, 2.0 eq), and the reaction was heated toreflux for 16 has progress of the reaction was monitored by TLC (mobilephase: 10% MeOH in DCM, visualization by UV). The crude reaction mixturewas concentrated under reduced pressure and the product was trituratedwith EtOAc (15 mL) to afford product1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) azepan-1-iumbromide (0.12 g). MS (ESI): m/z 351.2 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ10.04 (s, 1H), 7.71-7.62 (m, 2H), 7.60-7.48 (m, 3H), 7.20-7.03 (m, 3H),4.93 (s, 2H), 4.15 (s, 2H), 3.86-3.70 (m, 2H), 3.66-3.42 (m, 2H), 2.23(s, 6H), 1.98 (br d, J=4.8 Hz, 4H), 1.67 (br s, 4H).

4. Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidin-1-ium bromide

Synthesis of Intermediate N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)acetamide

Synthesis of intermediate 2-bromo-N-(2,6-dimethylphenyl) acetamide wasas described above for Compound 2. To a stirred solution of2-bromo-N-(2,6-dimethylphenyl) acetamide (1.2 g, 5.0 mmol, 1.0 eq) inEtOAc (40 mL) was added piperidine (0.851 g, 10.0 mmol, 2.0 eq), and thereaction mixture was stirred for 16 h at 75° C. as the progress of thereaction was monitored by TLC (mobile phase: 50% EtOAc in petroleumether, visualization by UV). The reaction mixture was diluted with EtOAc(20 mL) and washed with water (2×50 ml). The organic extracts werecombined, dried over Na₂SO₄, filtered and concentrated under reducedpressure to afford crude product, which was triturated with n-pentane(25 mL) and dried under vacuo to afford productN-(2,6-dimethylphenyl)-2-(piperidin-1-yl) acetamide (1.05 g). MS (ESI):m/z 247.36 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃-d) δ ppm 8.81 (1H, br s),7.04-7.11 (3H, m), 3.14 (2H, s), 2.62 (4H, br s), 2.24 (6H, s), 1.65(4H, quin, J=5.61 Hz), 1.44-1.54 (2H, m).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidin-1-ium bromide

To a stirred solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)acetamide (0.15 g, 0.6 mmol, 1.0 eq) in ACN (3 mL) was added benzylbromide (0.205 g, 1.2 mmol, 2.0 eq), and the reaction mixture wasstirred for 16 h at 80° C. in a sealed tube as progress of the reactionwas monitored by TLC (mobile phase: 10% MeOH in DCM, visualization byUV). The reaction mixture was allowed to cool to room temperature andconcentrated under reduced pressure to afford crude product, which wastriturated with EtOAc (15 mL) and dried under vacuo to afford product1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidin-1-iumbromide (0.15 g). MS (ESI): m/z 337.2 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δppm 10.07 (1H, s), 7.49-7.66 (5H, m), 7.07-7.21 (3H, m), 4.96 (2H, s),4.21 (2H, s), 3.65 (2H, br d, J=12.50 Hz), 3.50 (2H, dt, J=12.93, 6.69Hz), 2.21 (6H, s), 1.91-2.05 (4H, m), 1.64-1.76 (1H, m), 1.55 (1H, dt,J=13.76, 7.04 Hz).

5. Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)pyrrolidin-1-ium bromide

Synthesis of Intermediate N-(2,6-dimethylphenyl)-2-(pyrrolidin-1-yl)acetamide

Synthesis of intermediate 2-bromo-N-(2,6-dimethylphenyl) acetamide wasas described above for Compound 2. To a stirred solution of2-bromo-N-(2,6-dimethylphenyl)acetamide (1.2 g, 5.0 mmol, 1.0) in EtOAc(40 mL) was added pyrrolidine (0.533 g, 7.5 mmol, 1.5 eq), and thereaction mixture was stirred for 3 h at 75° C. as the progress of thereaction was monitored by TLC (mobile phase: 50% EtOAc in petroliumether, visualization by UV). The reaction mixture was diluted with EtOAc(25 mL), washed with water (50 mL), dried over NaSO4, filtered andconcentrated under reduced pressure to affordN-(2,6-dimethylphenyl)-2-(pyrrolidin-1-yl) acetamide (1.0 g). MS (ESI):m/z 233.44 [M+H]⁺. ¹H NMR (400 MHz, CDCl3-d) δ ppm 8.63 (1H, br s),7.00-7.15 (3H, m), 3.35 (2H, s), 2.69-2.84 (4H, m), 2.24 (6H, s),1.80-1.93 (4H, m).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)pyrrolidin-1-ium bromide

To a stirred solution of N-(2,6-dimethylphenyl)-2-(pyrrolidin-1-yl)acetamide (0.15 g, 0.6 mmol, 1.0 eq) in ACN (5 mL) added benzyl bromide(0.205 g, 1.2 mmol, 2.0 eq), and the reaction mixture was stirred for 16h at 80° C. in a sealed tube as progress of the reaction was monitoredby TLC (mobile phase: 10% MeOH in DCM, visualization by UV). Thereaction cooled to room temperature and was concentrated under reducedpressure to afford crude product, which was triturated with EtOAc (15mL) and dried under vacuo to afford1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) pyrrolidin-1-iumbromide (0.14 g). MS (ESI): m/z 323.2 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δppm 2.11-2.29 (10H, m), 3.60-3.74 (2H, m), 3.76-3.88 (2H, m), 4.20 (2H,s), 4.83 (2H, s), 7.08-7.18 (3H, m), 7.50-7.66 (5H, m), 9.97 (1H, s).

6. Synthesis of1-benzyl-1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)piperidin-1-iumbromide

Synthesis of Intermediate 2-bromobutanoyl chloride

Thionyl chloride (150 mL) was added to 2-bromobutanoic acid (25 g, 149.7mmol, 1.0 eq) at 0° C. and the resulting solution was subsequentlystirred at 80° C. for 2 h. The reaction mixture was cooled to RT andconcentrated under reduced pressure to afford crude 2-bromobutanoylchloride (27.7 g, 99.5%) as a brown residue which was taken to next stepimmediately without further purification. ¹H NMR (400 MHz, CDCl₃) δ4.52-4.44 (m, 1H), 2.29-2.16 (m, 1H), 2.15-2.02 (m, 1H), 1.10 (t, J=7.3Hz, 3H).

Synthesis of Intermediate 2-bromo-N-(2,6-dimethylphenyl) butanamide

A solution of 2,6-dimethylaniline (15 g, 121.18 mmol, 1.0 eq) andpyridine (15 mL, 189.6 mmol, 1.5 eq) in DCM (400 mL) was cooled in anice bath to 0° C. To this mixture was slowly added a solution of2-bromobutanoyl chloride (27.5 g, 148.6 mmol, 1.2 eq) in DCM (50 mL) andthe resulting mixture was allowed to warm to room temperature whilestirring for 2 h. The reaction solution was adjusted to pH 5 to 6 with 2N HCl and extracted with DCM (2×200 mL). The combined organic extractswere washed with water (250 mL) and brine (200 mL), dried over Na₂SO₄,filtered and concentrated under reduced pressure. The resulting crudeproduct was triturated with n-pentane (150 mL) to afford2-bromo-N-(2,6-dimethylphenyl)butanamide (30 g). MS (ESI): m/z 272.13[M+2]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.69 (s, 1H), 7.15-7.02 (m, 3H),4.51 (t, J=7.3 Hz, 1H), 2.22-2.04 (m, 7H), 2.03-1.91 (m, 1H), 0.98 (t,J=7.3 Hz, 3H).

Synthesis of Intermediate N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide

To a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)butanamide (50 g,259.1 mmol, 1.0 eq) in toluene (1 L) was added piperidine (54 mL, 544.1mmol, 2.1 eq) at RT. The reaction mixture was stirred at 110° C. for 16h as progress of the reaction was monitored by TLC (mobile phase: 30%EtOAc in hexane, visualization by UV). The reaction mixture wasconcentrated under reduced pressure to afford crude product, which wastriturated with n-pentane (500 mL) to affordN-(2,6-dimethylphenyl)-2-(piperidin-1-yl) butanamide (55 g) MS (ESI):m/z 275.27 [M+H]⁺. 1H NMR (400 MHz, DMSO-d₆) δ 9.16 (s, 1H), 7.06 (s,3H), 3.06 (dd, J=5.7, 8.6 Hz, 1H), 2.60 (br t, J=4.8 Hz, 4H), 2.16 (s,6H), 1.79-1.59 (m, 2H), 1.51 (br s, 4H), 1.44-1.35 (m, 2H), 0.93 (t,J=7.5 Hz, 3H).

Synthesis of 1-benzyl-1-(1-((2,6-dimethylphenyl) amino)-1-oxobutan-2-yl)piperidin-1-ium bromide

To a stirred solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide (1.5 g, 5.5 mmol, 1.0 eq) in ACN (25.0 mL) was added benzylbromide (1.872 g, 10.9 mmol, 2.0 eq) and the reaction mixture was heatedto 80° C. for 16 h as progress of the reaction was monitored by TLC(mobile phase: 10% MeOH in DCM, visualization by UV). The reactionmixture was concentrated under reduced pressure and the crude productwas triturated with EtOAc (3×10 mL) to afford1-benzyl-1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)piperidin-1-iumbromide (1.2 g) MS (ESI): m/z 365.4 [M].+. 1H NMR (400 MHz, DMSO-d₆) δ10.36 (s, 1H), 7.57-7.54 (m, 5H), 7.17-7.11 (m, 3H), 5.41 (d, 1H), 4.62(d, 1H), 4.39 (bs, 1H), 3.72 (bs, 2H), 3.22 (bs, 2H), 2.43-2.39 (m, 1H),2.15-2.07 (m, 9H), 1.96-1.95 (m, 2H), 1.61-1.45 (m, 2H), 1.18 (t, 3H).

7. Synthesis of Compounds 7A-27A: Table 5

Table 5 provides additional representative examples of the inventionwhich were prepared from intermediateN-(2,6-dimethylphenyl)-2-(piperidin-1-yl) butanamide and the appropriatealkyl halide according to the described methods for the synthesis ofcompound 6.

TABLE 5 MS (ESI): Compound # Structure m/z 1H NMR (400 MHz, DMSO-D6) δ 7A

383.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.17 (3 H, br t, J = 6.80Hz), 1.35-1.69 (2 H, m), 1.81-2.03 (3 H, m), 2.05-2.27 (8 H, m),2.31-2.47 (1 H, m), 3.10-3.25 (2 H, m), 3.57-3.89 (2 H, m), 4.33-4.55 (1H, m), 4.63 (1 H, br d, J = 13.59 Hz), 5.40 (1 H, br d, J = 13.59 Hz),7.04-7.22 (3 H, m), 7.38 (2 H, br t, J = 8.66 Hz), 7.61 (2 H, dd, J =8.33, 5.48 Hz), 10.36 (1 H, br s)  8A

383.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.18 (3 H, br t, J = 7.02Hz), 1.47 (1 H, br s), 1.59 (1 H, br s), 1.80- 2.04 (3 H, m), 2.08-2.16(1 H, m), 2.21 (8 H, s), 3.23 (2 H, br d, J = 16.66 Hz), 3.64-3.85 (2 H,m), 4.36-4.72 (2 H, m), 5.42 (1 H, br d, J = 13.15 Hz), 7.06-7.23 (3 H,m), 7.33-7.51 (3 H, m), 7.51-7.65 (1 H, m), 10.39 (1 H, s)  9A

383.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.11-1.30 (3 H, m), 1.42 (1H, br d, J = 1.75 Hz), 1.62 (1 H, br d, J = 11.40 Hz), 1.77-2.06 (3 H,m), 2.10-2.43 (9 H, m), 3.03-3.24 (2 H, m), 3.61-3.90 (2 H, m),4.54-4.80 (2 H, m), 5.63 (1 H, br d, J = 12.93 Hz), 7.03-7.25 (3 H, m),7.32-7.50 (2 H, m), 7.58-7.72 (2 H, m), 10.43 (1 H, s) 10A

399.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.11-1.23 (3 H, m), 1.42-1.67(2 H, m), 1.80-2.03 (3 H, m), 2.10- 2.29 (8 H, m), 2.31-2.46 (1 H, m),3.21 (2 H, br s), 3.57-3.88 (2 H, m), 4.27-4.52 (1 H, m), 4.63 (1 H, brd, J = 13.59 Hz), 5.40 (1 H, br d, J = 13.59 Hz), 7.04-7.22 (3 H, m),7.50-7.67 (4 H, m), 10.35 (1 H, s) 11A

399.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.18 (3 H, br t, J = 6.91Hz), 1.38- 1.65 (2 H, m), 1.79-2.01 (2 H, m), 2.03-2.28 (8 H, m), 2.40(1 H, br dd, J = 12.93, 7.02 Hz), 3.05-3.26 (2 H, m), 3.76 (2 H, br d, J= 6.14 Hz), 4.31- 4.57 (1 H, m), 4.63 (1 H, br d, J = 13.37 Hz), 5.41 (1H, br d, J = 13.37 Hz), 7.06-7.23 (3 H, m), 7.44-7.61 (2 H, m),7.62-7.71 (2 H, m), 10.36 (1 H, s) 12A

399.1 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.24 (3 H, br t, J = 6.91Hz), 1.29- 1.41 (1 H, m), 1.65 (1 H, br d, J = 12.50 Hz), 1.81 (2 H, brs), 1.92- 2.10 (1 H, m), 2.11-2.29 (7 H, m), 2.31-2.46 (2 H, m), 3.03 (1H, br t, J = 12.28 Hz), 3.14-3.26 (1 H, m), 3.77 (1 H, br d, J = 13.15Hz), 3.98 (1 H, br d, J = 12.93 Hz), 4.65 (1 H, br d, J = 13.37 Hz),4.84 (1 H, br d, J = 9.65 Hz), 5.82 (1 H, br d, J = 13.15 Hz), 7.16 (3H, s), 7.46-7.55 (1 H, m), 7.59 (1 H, br t, J = 7.23 Hz), 7.62- 7.71 (1H, m), 7.76 (1 H, br d, J = 7.23 Hz), 10.41 (1 H, br s) 13A

390.3 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.17 (3 H, br t, J = 6.58Hz), 1.39- 1.68 (2 H, m), 1.79-2.04 (3 H, m), 2.08-2.28 (8 H, m),2.35-2.46 (1 H, m), 3.09-3.26 (2 H, m), 3.64-3.88 (2 H, m), 4.30-4.60 (1H, m), 4.72 (1 H, br d, J = 13.37 Hz), 5.49 (1 H, br d, J = 13.37 Hz),7.04-7.25 (3 H, m), 7.76 (2 H, d, J = 8.11 Hz), 8.03 (2 H, d, J = 8.11Hz), 10.35 (1 H, s) 14A

390.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.20 (3 H, br t, J = 6.80Hz), 1.36- 1.52 (1 H, m), 1.59 (1 H, br s), 1.77- 2.03 (3 H, m),2.07-2.17 (1 H, m), 2.22 (6 H, s), 2.28-2.45 (2 H, m), 3.04-3.26 (2 H,m), 3.77 (2 H, br s), 4.39-4.76 (2 H, m), 5.46 (1 H, br d, J = 13.15Hz), 7.06-7.25 (3 H, m), 7.67-7.81 (1 H, m), 7.90 (1 H, br d, J = 7.89Hz), 7.99-8.13 (2 H, m), 10.39 (1 H, br s) 15A

390.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.18-1.48 (4 H, m), 1.65 (1H, br d, J = 11.40 Hz), 1.82 (2 H, br d, J = 14.03 Hz), 1.94-2.11 (1 H,m), 2.25 (7 H, s), 2.33-2.47 (2 H, m), 3.07-3.24 (2 H, m), 3.76 (1 H, brd, J = 11.18 Hz), 3.97 (1 H, br d, J = 11.40 Hz), 4.76 (1 H, br d, J =13.81 Hz), 4.88 (1 H, br d, J = 5.26 Hz), 5.75 (1 H, br d, J = 13.59Hz), 7.16 (3 H, br s), 7.78 (1 H, br d, J = 5.26 Hz), 7.88 (2 H, br s),8.05 (1 H, br d, J = 7.45 Hz), 10.45 (1 H, br s) 16A

379.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.17 (3 H, br t, J = 6.80Hz), 1.32- 1.66 (2 H, m), 1.82-2.03 (3 H, m), 2.06-2.27 (8 H, m), 2.38(4 H, s), 3.05-3.27 (2 H, m), 3.57-3.89 (2 H, m), 4.25-4.47 (1 H, m),4.57 (1 H, br d, J = 13.37 Hz), 5.37 (1 H, br d, J = 13.37 Hz),7.00-7.24 (3 H, m), 7.28-7.51 (4 H, m), 10.38 (1 H, s) 17A

379.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.18 (3 H, br t, J = 6.80Hz), 1.43 (1 H, br d, J = 1.32 Hz), 1.58 (1 H, br s), 1.83-2.03 (3 H,m), 2.05-2.29 (8 H, m), 2.33-2.43 (4 H, m), 3.05- 3.27 (2 H, m), 3.74 (2H, br d, J = 7.02 Hz), 4.31-4.62 (2 H, m), 5.38 (1 H, br d, J = 12.93Hz), 7.06-7.20 (3 H, m), 7.28-7.49 (4 H, m), 10.38 (1 H, s) 18A

379.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.25 (4 H, br t, J = 7.23Hz), 1.66 (1 H, br d, J = 12.28 Hz), 1.79 (2 H, br t, J = 12.72 Hz),1.93-2.10 (1 H, m), 2.11-2.30 (7 H, m), 2.30-2.48 (5 H, m), 2.85 (1 H,br t, J = 12.50 Hz), 3.12- 3.28 (1 H, m), 3.49-3.65 (1 H, m), 3.97 (1 H,br d, J = 12.72 Hz), 4.52 (1 H, br d, J = 13.37 Hz), 4.83 (1 H, br d, J= 10.08 Hz), 5.74 (1 H, br d, J = 13.37 Hz), 7.09-7.27 (3 H, m),7.27-7.40 (2 H, m), 7.40-7.47 (1 H, m), 7.52 (1 H, br d, J = 7.45 Hz),10.43 (1 H, s) 19A

393.3 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.08-1.31 (6 H, m), 1.36-1.67(2 H, m), 1.81-2.06 (3 H, m), 2.06- 2.32 (8 H, m), 2.33-2.44 (1 H, m),2.68 (2 H, br d, J = 7.23 Hz), 2.99- 3.26 (2 H, m), 3.58-3.88 (2 H, m),4.29-4.69 (2 H, m), 5.37 (1 H, br d, J = 12.93 Hz), 7.14 (3 H, br s),7.29- 7.54 (4 H, m), 10.39 (1 H, br s) 20A

393.3 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.08-1.29 (6 H, m), 1.35-1.52(1 H, m), 1.59 (1 H, br d, J = 3.73 Hz), 1.82-2.04 (3 H, m), 2.07-2.27(8 H, m), 2.40 (1 H, br dd, J = 12.93, 7.67 Hz), 2.68 (2 H, q, J = 7.45Hz), 3.08- 3.27 (2 H, m), 3.61-3.86 (2 H, m), 4.30-4.52 (1 H, m), 4.58(1 H, br d, J = 13.37 Hz), 5.39 (1 H, br d, J = 13.15 Hz), 7.03-7.24 (3H, m), 7.27-7.51 (4 H, m), 10.36 (1 H, s) 21A

393.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.14 (3 H, t, J = 7.45 Hz),1.26 (4 H, br t, J = 7.34 Hz), 1.66 (1 H, br d, J = 12.50 Hz), 1.78 (2H, br t, J = 15.13 Hz), 1.95-2.12 (1 H, m), 2.13-2.29 (7 H, m),2.30-2.48 (2 H, m), 2.60- 2.90 (3 H, m), 3.16-3.27 (1 H, m), 3.53 (1 H,br d, J = 13.15 Hz), 3.98 (1 H, br d, J = 12.72 Hz), 4.50 (1 H, br d, J= 13.37 Hz), 4.87 (1 H, br d, J = 10.52 Hz), 5.81 (1 H, br d, J = 13.37Hz), 7.10-7.25 (3 H, m), 7.28-7.39 (1 H, m), 7.40-7.58 (2 H, m), 10.45(1 H, br s) 22A

395.4 [M]+ 1H NMR (500 MHz, DMSO-d6) δ ppm 1.15 (3 H, br s), 1.36-1.66(2 H, m), 1.85-2.26 (11 H, m), 2.31-2.45 (1 H, m), 3.08-3.25 (2 H, m),3.82 (3 H, s), 4.56 (1 H, br d, J = 13.43 Hz), 5.33 (1 H, br d, J =13.73 Hz), 7.02- 7.20 (5 H, m), 7.45 (2 H, d, J = 8.85 Hz), 10.33 (1 H,s) 23A

395.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.18 (3 H, br s), 1.35-1.69(2 H, m), 1.77-2.29 (11 H, m), 2.41 (1 H, br dd, J = 11.84, 7.45 Hz),3.06-3.28 (2 H, m), 3.62-3.91 (5 H, m), 4.32- 4.66 (2 H, m), 5.37 (1 H,br d, J = 13.15 Hz), 7.03-7.22 (6 H, m), 7.46 (1 H, br t, J = 7.89 Hz),10.40 (1 H, s) 24A

395.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.18 (3 H, br t, J = 7.02Hz), 1.36 (1 H, br d, J = 3.95 Hz), 1.62 (1 H, br d, J = 13.15 Hz),1.75-2.01 (3 H, m), 2.04-2.43 (9 H, m), 2.94-3.23 (2 H, m), 3.61-3.91 (5H, m), 4.47 (1 H, br d, J = 13.15 Hz), 4.54-4.69 (1 H, m), 5.51 (1 H, brd, J = 12.06 Hz), 7.00- 7.27 (5 H, m), 7.42-7.61 (2 H, m), 10.34 (1 H,s) 25A

386.4 [M]+ 1H NMR (500 MHz, DMSO-d6) δ ppm 1.12 (4 H, br t, J = 7.17Hz), 1.38- 1.71 (3H, m), 1.82-2.33 (16 H, m), 2.73 (4 H, br s),2.91-3.08 (1 H, m), 3.74-4.33 (3 H, m), 4.60-5.04 (2 H, m), 5.44-5.71 (1H, m), 6.40-6.65 (1 H, m), 6.69-6.91 (1 H, m), 6.97- 7.22 (3 H, m), 7.92(1 H, br s), 10.38- 10.68 (1 H, m) 26A

371.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.15 (3 H, br t, J = 7.23Hz), 1.56 (2 H, br s), 1.96 (2 H, br s), 2.02- 2.27 (9 H, m), 2.29-2.46(1 H, m), 3.30 (2 H, s), 3.59 (1 H, br s), 3.84 (1 H, br s), 4.31 (1 H,br d, J = 5.48 Hz), 4.65 (1 H, br d, J = 13.81 Hz), 5.34 (1 H, br d, J =13.81 Hz), 7.05-7.20 (3 H, m), 7.22-7.33 (1 H, m), 7.76 (1 H, br dd, J =4.82, 2.85 Hz), 7.93 (1 H, d, J = 1.75 Hz), 10.39 (1 H, s) 27A

370.2 [M]+ 1H NMR (400 MHz, DMSO-d6) δ ppm 1.00-1.27 (3 H, m), 1.42-1.74(2 H, m), 1.81-2.28 (12 H, m), 2.28- 2.42 (2 H, m), 3.38-3.67 (3 H, m),3.84-4.10 (1 H, m), 4.22 (1 H, br dd, J = 5.70, 2.85 Hz), 4.78 (1 H, brd, J = 13.81 Hz), 5.41 (1 H, br d, J = 14.03 Hz), 6.60 (1 H, br s), 7.13(3 H, br s), 10.39 (1 H, br s)

28. Synthesis of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-(2-hydroxybenzyl) piperidin-1-ium2,2,2-trifluoroacetate

To a stirred solution of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-(2-methoxybenzyl) piperidin-1-ium bromide(compound 24, 0.1 g, 0.21 mmol, 1.0 eq) in DMC (2 mL) was added borontribromide (1M in DCM) (4 mL, 4.0 mmol, 20 eq) at 0° C., and thereaction mixture was stirred for 16 h at room temperature as progress ofthe reaction was monitored by TLC (mobile phase: 10% MeOH in DCM,visualization by UV). The reaction mixture was concentrated underreduced pressure to afford crude product which was purified bypreparative HPLC (mobile phase A: 0.1% TFA (aq.); mobile phase B: ACN;column: Synergy Polar 250×21 mm, 4.7u (Phenomenex Inc.); flow: 18mL/min, method: 0/15, 2/15, 10/50, 15/80, 15.2/98, 18/98, 18.2/15,22/15; temperature: ambient) Pure fractions were lyophilized to affordproduct 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-(2-hydroxybenzyl) piperidin-1-ium2,2,2-trifluoroacetate (0.056 g). MS (ESI): m/z 381.4[M]+. 1H NMR (500MHz, DMSO-d6) δ ppm 1.17 (3H, br t, J=7.02 Hz), 1.32-1.47 (1H, m),1.55-1.67 (1H, m), 1.76-2.01 (3H, m), 2.05-2.19 (2H, m), 2.25-2.3 (6H,m), 2.30-2.41 (1H, m), 2.99-3.26 (1H, m), 3.64-3.85 (2H, m), 4.35-4.61(2H, m), 5.41-5.50 (1H, m), 6.88-6.95 (1H, m), 6.99 (1H, d, J=8.24 Hz),7.10-7.19 (3H, m), 7.31-7.45 (2H, m), 10.34 (2H, br d, J=10.07 Hz).

29. Synthesis of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-(3-hydroxybenzyl) piperidin-1-ium carbonate

To a stirred solution of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-(3-methoxybenzyl) piperidin-1-ium bromide(compound 23, 0.06 g, 0.1 mmol, 1 eq) in DCM (2 mL) was added borontribromide (1 M in DCM) (2 mL) at 0° C., and the reaction mixture wasstirred for 16 h at room temperature as progress was monitored by TLC(mobile phase: 10% MeOH in DCM, visualization by UV). The reactionmixture was concentrated under reduced pressure to afford crude product,which was purified by reverse phase preparative-HPLC (mobile phase (A):10 mM ammonium bicarbonate (aq.); mobile phase (B): 100% CAN; column:X-select C18 (19*250) 5u, method: 0/35, 2/40, 20/40; flow: 18 ml/min.The combined pure fractions were lyophilized to afford product1-(1-((2,6-dimethylphenyl) amino)-1-oxobutan-2-yl)-1-(3-hydroxybenzyl)piperidin-1-ium carbonate (0.02 g). MS (ESI): m/z 381.38 [M]⁺. ¹H NMR(500 MHz, DMSO-d₆) δ ppm 1.11 (3H, br s), 1.31-1.62 (2H, m), 1.76-2.25(12H, m), 2.93-3.13 (1H, m), 3.43-3.54 (3H, m), 3.60-3.76 (1H, m),3.92-4.17 (2H, m), 4.21-4.39 (1H, m), 5.29-5.63 (1H, m), 6.67-6.85 (4H,m), 6.91 (2H, br s), 7.19 (1H, br t, J=7.78 Hz).

30. Synthesis of 1-benzyl-1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl) azepan-1-ium bromide

Synthesis of Intermediate 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)butanamide

Synthesis of intermediate 2-bromo-N-(2,6-dimethylphenyl) butanamide wasas described above for Compound 6. To a stirred solution of2-bromo-N-(2,6-dimethylphenyl) butanamide (0.5 g, 1.9 mmol, 1.0 eq) inACN (10 mL) was added K₂CO₃ (0.393 g, 2.85 mmol, 1.5 eq) and azepane(0.376 g, 3.8 mmol, 2.0 eq), and the reaction mixture was stirred for 16h at 75° C. as progress of the reaction was monitored by TLC (mobilephase: 10% MeOH in DCM, visualization by UV). The crude reaction mixturewas quenched with water (10 mL) and extracted EtOAc (2×15 mL). Thecombined organic extracts were dried over Na₂SO₄, filtered andconcentrated under reduced pressure to afford intermediate2-(azepan-1-yl)-N-(2,6-dimethylphenyl) butanamide (0.20 g). MS (ESI):m/z 289.33 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃-d) δ ppm 1.00-1.16 (3H, m),1.57-1.86 (12H, m), 1.95-2.13 (2H, m), 2.18-2.29 (8H, m), 2.72-2.97 (4H,m), 3.24 (1H, br t, J=6.72 Hz), 3.46-3.55 (2H, m), 7.03-7.15 (4H, m),8.75 (1H, br s).

Synthesis of 1-benzyl-1-(1-((2,6-dimethylphenyl) amino)-1-oxobutan-2-yl)azepan-1-ium bromide

To a stirred solution of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)butanamide (0.2 g, 0.7 mmol, 1.0 eq) in ACN (5 mL) was added benzylbromide (0.459 g, 2.85 mmol, 1.5 eq) and the reaction was stirred for 16h at 75° C. as progress was monitored by TLC (mobile phase: 10% MeOH inDCM, visualization by UV). The reaction mixture was cooled to roomtemperature and concentrated under reduced pressure to afford crudeproduct, which was triturated with EtOAc (20 mL) and dried under vacuoto afford 1-benzyl-1-(1-((2,6-dimethylphenyl) amino)-1-oxobutan-2-yl)azepan-1-ium bromide (0.118 g). MS (ESI): m/z 379.52 [M]+. 1H NMR (500MHz, DMSO-d6) δ ppm 1.16 (3H, t, J=7.32 Hz), 1.26-1.51 (4H, m), 1.60(2H, br s), 1.77-1.94 (2H, m), 2.10-2.31 (7H, m), 2.34-2.48 (1H, m),3.56-3.81 (4H, m), 4.26 (1H, br d, J=10.68 Hz), 4.66 (1H, d, J=13.12Hz), 5.21 (1H, d, J=13.12 Hz), 7.06-7.22 (3H, m), 7.45-7.61 (3H, m),7.61-7.69 (2H, m), 10.42 (1H, s).

31. Synthesis of1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)azocan-1-iumbromide

Synthesis of Intermediate 2-(azocan-1-yl)-N-(2,6-dimethylphenyl)acetamide

To a stirred solution of 2-bromo-N-(2,6-dimethylphenyl) acetamide (500mg, 2.065 mmol) in ACN (5.0 mL) was added K₂CO₃ (713 mg, 5.159 mmol)followed by azocane (467 mg, 4.125 mmol) and the resulting reactionmixture was stirred for 16 h at 80° C. as progress of the reaction wasmonitored by TLC (5% MeOH in DCM, visualization by UV). The crudereaction mixture was poured into ice-cold water (40 mL) and extractedwith EtOAc (2×50 mL). The combined organic extracts were washed withbrine solution (50 mL), dried over anhydrous Na₂SO₄, and subsequentlyconcentrated under reduced pressure to afford2-(azocan-1-yl)-N-(2,6-dimethylphenyl) acetamide (480 mg) as an offwhite solid. Mass (ESI): m/z 275.1 [M+H]+. 1H NMR (400 MHz,CHLOROFORM-d) δ ppm 8.74 (br s, 1H), 7.02-7.16 (m, 3H), 3.34 (s, 2H),2.74-2.88 (m, 4H), 2.25 (s, 6H), 1.53-1.80 (m, 10H).

Synthesis of1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)azocan-1-iumbromide

To a stirred solution of 2-(azocan-1-yl)-N-(2,6-dimethylphenyl)acetamide (200 mg, 0.728 mmol) in ACN (2.0 mL) was added benzyl bromide(240 mg, 1.403 mmol) and the resulting reaction mixture was stirred for16 h at 90° C. as progress of the reaction was monitored by TLC (5% MeOHin DCM, visualization by UV). The reaction mixture was concentratedunder reduced pressure to afford crude product which was triturated witha 1:1 mixture of EtOAc:Et2O (3×50 mL) to afford1-benzyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl) azocan-1-iumbromide as a pale brown solid. Mass (ESI): m/z 365.54 [M]+. 1H NMR (400MHz, DMSO-d6) δ ppm 10.02 (s, 1H), 7.46-7.70 (m, 5H), 7.05-7.22 (m, 3H),4.89 (s, 2H), 4.07 (s, 2H), 3.64-3.76 (m, 2H), 3.48-3.62 (m, 2H), 2.23(s, 6H), 1.94-2.14 (m, 4H), 1.50-1.83 (m, 6H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3,3-difluoropiperidin-1-ium bromide

Synthesis of 2-(3,3-difluoropiperidin-1-yl)-N-(2,6-dimethylphenyl)acetamide

To a stirred solution of 3,3-difluoropiperidine hydrochloride salt(0.371 g, 0.309 mmol) in ACN (25 mL) at 0° C. was added TEA (0.626 g,6.195 mmol) at 0° C. After stirring for 10 minutes at 0° C.,2-bromo-N-(2,6-dimethylphenyl) acetamide (0.5 g, 2.065 mmol) was addedat 0° C. The resulting reaction mixture was stirred for 16 h at roomtemperature as progress of the reaction was monitored by TLC (30% EtOAcin Pet Ether, visualization by UV). The reaction mixture was dilutedwith water (10 mL) and extracted with ETOAc (2×25 mL).

The combined organic layers were concentrated under reduced pressure toafford crude product which was purified by normal phase flashchromatography (5% MeOH in DCM) to afford2-(3,3-difluoropiperidin-1-yl)-N-(2,6-dimethylphenyl) acetamide (0.4 g)as a white solid. MS (ESI): m/z 283.24 [M+H]⁺. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 8.58 (s, 1H), 7.07-7.13 (m, 3H), 3.27 (s, 2H), 2.86(t, 2H), 2.50 (d, 2H), 2.23 (s, 6H), 1.86-2.04 (m, 4H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3,3-difluoropiperidin-1-ium bromide

To a solution of 2-(3,3-difluoropiperidin-1-yl)-N-(2,6-dimethylphenyl)acetamide (0.2 g, 0.708 mmol) in ACN (10 mL) was added benzyl bromide(0.145 g, 0.850 mmol) at room temperature in a sealed tube. Theresulting reaction mixture was heated to 75° C. for 16 h as progress ofthe reaction was monitored by TLC (5% MeOH in DCM, visualization by UV).The reaction mixture was diluted with water (25 mL) and extracted with10% methanol in DCM (2×100 mL). The combined organic layers wereconcentrated under reduced pressure to afford crude product which wastriturated with ethyl acetate (10 mL) to afford1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3,3-difluoropiperidin-1-ium bromide (0.060 g) as anoff white solid. MS (ESI): m/z 373.1 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δppm 10.10 (s, 1H), 7.56-7.62 (m, 5H), 7.07-7.18 (m, 3H), 5.16 (d, 1H),5.01 (d, 1H), 4.31-4.40 (m, 2H), 3.98-4.18 (m, 2H), 3.61-3.68 (m, 2H),2.11-2.48 (m, 10H).

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-((phenyl-d5) methyl) azepan-1-ium chloride

Synthesis of 1-(chloromethyl) benzene-2,3,4,5,6-d5

To a stirred solution of benzene-d6 (5 g, 59.41 mmol) in Conc. HCl (15mL) was added paraformaldehyde (5.3522 g, 178.23 mmol) at roomtemperature, and the reaction mixture was stirred for 48 h at 80° C. insealed tube as progress of the reaction was monitored by 1H NMR Thereaction mixture was extracted with Et₂O (2×25 mL) and the combinedorganic extracts washed with saturated sodium bicarbonate solution (50mL), dried over anhydrous sodium sulphate and concentrated under reducedpressure at 25° C. to afford 1-(chloromethyl) benzene-2,3,4,5,6-d5 (1.6g) as a colourless liquid. H NMR (400 MHz, CDCl₃) δ ppm 4.6 (s, 2H).

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-((phenyl-d5) methyl) azepan-1-ium chloride

To a stirred solution of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)acetamide (1.5 g, 5.76 mmol) in ACN (15 mL) was added 1-(chloromethyl)benzene-2,3,4,5,6-d5 (1.592 g, 12.1 mmol) and the resulting reactionmixture was stirred at 80° C. for 16 h in a sealed tube as progress ofthe reaction was monitored by TLC (10% MeOH in DCM, Visualization byUV). The reaction mixture was cooled to room temperature, was filteredand the precipitated solid was washed with EtOAc to afford1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)-1-((phenyl-d5) methyl)azepan-1-ium chloride (1.67 g) as a white solid. MS (ESI): m/z 356.36[M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.55 (s, 1H), 7.1-7.18 (m, 3H),4.95 (s, 2H), 4.2 (s, 2H), 3.72-3.81 (m, 1H), 3.5-3.6 (m, 1H), 2.25 (s,6H), 1.9-2.1 (m, 4H), 1.1-1.2 (m, 4H).

Synthesis of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-((1-methyl-1H-pyrazol-4-yl) methyl)piperidin-1-ium bromide

Synthesis of 4-(bromomethyl)-1-methyl-1H-pyrazole hydrogen bromide

To a stirred solution of (1-methyl-1H-pyrazol-4-yl) methanol (1.0 g,8.918 mmol) in AcOH (5 mL) was added 33% HBr in AcOH (12 mL) at rt. Thereaction mixture was heated at 100° C. for 16 h as progress of thereaction was monitored by TLC (10% MeOH in DCM, visualization by UV).The reaction mixture was concentrated under reduced pressure to afford4-(bromomethyl)-1-methyl-1H-pyrazole hydrogen bromide (1.5 g) as palebrown solid. ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.01 (s, 1H), 7.50 (s, 1H),5.51 (s, 2H), 3.81 (s, 3H).

Synthesis of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-((1-methyl-1H-pyrazol-4-yl) methyl)piperidin-1-ium bromide

To a solution of 4-(bromomethyl)-1-methyl-1H-pyrazole.hydrogenbromide(0.5 g, 1.953 mmol) in DMF (5 mL) was addedN-(2,6-dimethylphenyl)-2-(piperidin-1-yl) butanamide (0.783 g, 2.853mmol) and potassium carbonate (0.473 g, 3.427 mmol). The resultingreaction mixture was heated at 100° C. for 24 h in a sealed tube asprogress of the reaction was monitored by TLC (5% MeOH in DCM,visualization by UV). The reaction mixture was diluted with water (25mL) and extracted with 10% methanol in DCM (2×100 ml). The combinedorganic layer was concentrated under reduced pressure to afford crudeproduct which was purified by normal phase flash chromatography (5% MeOHin DCM) to afford 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutan-2-yl)-1-((1-methyl-H-pyrazol-4-yl) methyl)piperidin-1-ium bromide (0.120 g) as an off white solid. MS (ESI): m/z369.48 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.4 (s, 1H), 8.01 (s, 1H),7.64 (s, 1H), 7.10-7.14 (m, 3H), 5.17 (d, 1H), 4.48 (d, 1H), 4.1-4.3 (m,1 H), 3.90 (s, 3H), 3.8-3.9 (m, 1H), 3.20-3.45 (m, 2H), 2.30-2.36 (m,1H), 2.16 (s, 6H), 1.92-2.08 (m, 6H), 1.54 (d, 2H), 1.11-1.17 (m, 3H).

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(3-(methoxycarbonyl) benzyl) piperidin-1-iumbromide

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(3-(methoxycarbonyl) benzyl) piperidin-1-iumbromide

To a solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl) acetamide(200 mg, 0.812 mmol) in ACN (3 mL) was added methyl3-(bromomethyl)benzoate (241.6 mg, 1.055 mmol) at room temperature. Theresulting reaction mixture was stirred at 90° C. for 16 h as progress ofthe reaction was monitored by TLC (5% MeOH in DCM, Visualization: UV).The reaction mixture was cooled to room temperature and was concentratedunder reduced pressure to afford crude product which was triturated withEtOAc (3×10 mL) to afford pure 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(3-(methoxycarbonyl) benzyl) piperidin-1-iumbromide (57.5 mg) as an off white solid. MS (ESI): m/z 395.2 [M]⁺. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 10.09 (s, 1H), 8.18 (s, 1H), 8.13 (d, 1H),7.83-7.85 (m, 1H), 7.69-7.72 (m, 1H), 7.12-7.17 (m, 3H), 5.06 (s, 2H),4.22 (s, 2H), 3.88 (s, 3H), 3.52-3.65 (m, 2H), 3.47-3.50 (m, 2H), 2.21(s, 6H), 1.90-1.96 (m, 4H), 1.71-1.74 (m, 1H), 1.56-1.57 (m, 1H).

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(4-(methoxycarbonyl) benzyl) piperidin-1-iumbromide

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(4-(methoxycarbonyl) benzyl) piperidin-1-iumbromide

To a stirred solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)acetamide (0.4 g, 1.623 mmol) in ACN (5 mL) was added methyl4-(bromomethyl)benzoate (0.743 g, 3.247 mmol) and the resulting reactionmixture was stirred for 16 h at 80° C. as progress of the reaction wasmonitored by TLC (10% Methanol in DCM, visualisation: UV). The reactionmixture was allowed cool to room temperature and was concentrated underreduced pressure to afford crude product which was purified by refluxwith EtOAc (30 mLl) for 2 h at 70° C. Refluxed product was filtered andwashed with hot ethyl acetate (30 mL) to afford the(1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)-1-(4-(methoxycarbonyl)benzyl) piperidin-1-ium bromide (43 mg) as white solid. Mass (ESI): m/z395.3 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.06 (s, 1H), 8.09 (d, 2H),7.72 (d, 2H), 7.17-7.12 (m, 3H), 5.03 (s, 2H), 4.22 (s, 2H), 3.89 (s,3H), 3.67-3.64 (m, 2H), 3.54-3.49 (m, 2H), 2.20 (s, 6H), 2.0-1.97 (m,4H), 1.72-1.69 (m, 1H), 1.58-1.53 (m, 1H).

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(2-(methoxycarbonyl) benzyl) piperidin-1-iumbromide

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(2-(methoxycarbonyl) benzyl) piperidin-1-iumbromide

To a solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl) acetamide(0.500 g, 2.0296 mmol) in ACN (5.0 mL) was added methyl 2-(bromomethyl)benzoate (0.929 g, 4.0592 mmol) and the resulting reaction mixture washeated to 80° C. for 16 h as progress of the reaction was monitored byTLC (10% MeOH, Visualization: UV). The reaction mixture was concentratedunder reduced pressure to afford crude compound which was trituratedwith 3×10 mL of ethyl acetate followed by 2×10 mL of diethyl ether toafford pure 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(2-(methoxycarbonyl) benzyl) piperidin-1-iumbromide (250 mg) as an off white soli. MS (ESI): m/z 395.2 [M]⁺. ¹H NMR(400 MHz, DMSO-d6) δ ppm 10.11 (s, 1H), 8.01 (d, 1H), 7.69-7.76 (m, 3H),7.11-7.17 (m, 3H), 5.37 (s, 2H), 4.43-4.46 (m, 2H), 3.85 (s, 3H), 3.75(d, 2H), 3.17 (t, 2H), 2.20 (s, 6H), 1.96-2.07 (m, 2H), 1.79-1.83 (m,2H), 1.66-1.70 (m, 1H), 1.37-1.40 (m, 1H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) (methyl)amino)-2-oxoethyl) azepan-1-ium formate

Synthesis of 2-Iodo-N-(2,6-dimethylphenyl)-N-methyl acetamide

To a stirred solution of 2-chloro-N-(2,6-dimethylphenyl) acetamide (2.0g, 10.118 mmol) in THE (16 mL) was added NaH (60%) (0.607 g, 25.295mmol) at 0° C. and the mixture was stirred for 20 min at 0° C. Methyliodide (0.5 ml, 8.031 mmol) was added at 0° C. and resulting reactionmixture was stirred at room temperature 16 h as progress of the reactionwas monitored by TLC (10% EtOAc in Pet, Visualisation by UV). Thereaction mixture was poured into ice water (50 mL) and extracted withethyl acetate (2×50 mL). The combined organic extracts were washed withbrine solution (25 mL), dried over Na₂SO₄ and concentrated under reducedpressure to afford the crude product which was purified by columnchromatography (eluted with 20-30% of ethyl acetate in pet ether). Thecollected pure fractions were concentrated under reduced pressure toafford N-(2,6-dimethylphenyl)-2-iodo-N-methylacetamide (0.55 g) asyellow liquid. Mass (ESI): m/z 304.03 [M+H]⁺.

Synthesis of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)-N-methylacetamide

To a stirred solution of N-(2,6-dimethylphenyl)-2-iodo-N-methylacetamide(0.5 g, 1.649 mmol) in ACN (5.0 mL) was added K₂CO₃ (0.569 g, 4.123mmol) and azepane (0.327 ml, 3.298 mmol) at room temperature. Theresulting reaction mixture was stirred for 16 h at 90° C. as progress ofthe reaction was monitored by TLC (50% EtOAc in pet ether, visualizationby UV).

The reaction mixture was poured into ice water (25 mL) and extractedwith ethyl acetate (2×25 mL). The combined organic extracts were washedwith brine solution (25 mL), dried over Na₂SO₄ and concentrated underreduced pressure to afford the crude compound which was purified bynormal phase flash chromatography (eluted with 10%-50% EtOAc in petether) The collected pure fractions were concentrated under reducedpressure to afford 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)-N-methylacetamide (0.23 g). MS (ESI): m/z 275.49 [M+H]⁺.

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) (methyl)amino)-2-oxoethyl) azepan-1-iumformate

To a stirred solution of2-(azepan-1-yl)-N-(2,6-dimethylphenyl)-N-methylacetamide (0.1 g, 0.364mmol) in Acetonitrile (1.0 mL) was added (bromomethyl)benzene (0.124 g,0.728 mmol). The resulting reaction mixture was stirred for 16 h at 90°C. as progress of the reaction was monitored by TLC (10% MeOH in DCM,Visualisation: UV). The reaction mixture was concentrated under reducedpressure to afford the crude product which was purified by reverse phasePrep HPLC. (Column: X-select C18 (250*19) mm, 5u, Mobile phase A: 0.1%FA in water, Mobile phase B: ACN: MeOH, Flow: 13 ml/min, Solubility:Water+THF+ACN, Method (T % of B): 0/10, 2/10, 10/50, 13/50, 13.2/98,17/98, 17.2/10, 20/10, Temperature: Ambient). The collected purefractions were lyophilized to afford the product(1-benzyl-1-(2-((2,6-dimethylphenyl) (methyl) amino)-2-oxoethyl)azepan-1-ium formate (70 mg) as white gum. MS (ESI): m/z 365.3 [M]⁺. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 8.43 (s, 1H), 7.56-7.42 (m, 5H), 7.26-7.17(m, 3H), 4.95 (s, 2H), 4.41 (s, 1H), 3.91-3.85 (m, 1H), 3.62-3.50 (m,3H), 3.36 (s, 1H), 3.14 (d, 3H), 2.32-2.05 (s, 7H), 1.95-1.65 (m, 2H),1.46-1.23 (m, 5H).

Synthesis of: Synthesis of 1-benzyl-1-(1-(mesitylamino)-1-oxobutan-2-yl) piperidin-1-ium bromide

Synthesis of 2-bromo-N-mesitylbutanamide

To a stirred solution of 2,4,6-tri methyl aniline (2.0 g, 14.791 mmol)in DCM (20 mL) was added DIPEA (5.735 g, 44.373 mmol) at roomtemperature, followed by drop-wise addition of 2-bromo butanoyl chloride(3.291 g, 17.749 mmol) at 0° C. and the resulting reaction mixture wasstirred for 3 h as progress of the reaction was monitored by TLC (20%Ethyl acetate in Pet ether. Visualization: UV). The reaction mixture wasdiluted with water (100 mL) and extracted with EtOAc (2×100 mL). Thecombined organic extracts were washed with brine solution, dried overNa₂SO₄ and concentrated under reduced pressure to afford crude productwhich was purified by column chromatography (eluted with 10%-50% ofethyl acetate in pet ether). Pure fractions were combined andconcentrated to afford desired product 2-bromo-N-mesitylbutanamide (1.5g). MS (ESI): m/z 286.11 [M+2]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.60 (s,1H), 6.95 (s, 2H), 4.45 (t, 1H), 2.25 (s, 3H), 2.2 (s, 6H), 1.60-1.40(m, 2H), 1.10 (t, 3H).

Synthesis of N-mesityl-2-(piperidin-1-yl) butanamide

To a stirred solution of 2-bromo-N-mesityl butanamide (1.5 g, 5.278mmol) in Acetonitrile (30 ml) was added Potassium carbonate (2.18 g,15.834 mmol) and piperdine (0.943 g, 11.083 mmol) at room temperature.The resulting reaction was reflux for 16 h as progress of the reactionwas monitored by TLC (50% EtOAc/Hexane, Visualization: UV). The reactionmixture was diluted with water (50 mL) and extracted with EtOAc (3×20mL). The combined organic extracts were washed with brine solution,dried over Na₂SO₄ and concentrated under reduced pressure to affordcrude product which was triturated with ethyl acetate (40 mL) to affordpure N-mesityl-2-(piperidin-1-yl) butanamide (760 mg). MS (ESI): m/z289.43 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.07 (s, 1H), 6.86 (s,2H), 3.06-3.01 (t, 1H), 2.60-2.48 (m, 4H), 2.21 (s, 3H), 2.06 (s, 6H),1.80-1.25 (t, 8H), 0.95 (t, 3H).

Synthesis of 1-benzyl-1-(1-(mesitylamino)-1-oxobutan-2-yl)piperidin-1-ium bromide

To a stirred solution of N-mesityl-2-(piperidin-1-yl) butanamide (0.15g, 0.52 mmol) in Acetonitrile (1 mL) was added benzyl bromide (0.177 g,1.04 mmol). The resulting reaction mixture was stirred for 20 h at 80°C. as progress of the reaction was monitored by TLC (10% Methanol inDCM, Visualization: UV). The reaction mixture was concentrated underreduced pressure to afford crude product which was triturated with EtOAc(20 mL) to deliver desired product1-benzyl-1-(1-(mesitylamino)-1-oxobutan-2-yl) piperidin-1-ium bromide(194 mg) as a white solid. Mass (ESI): m/z 379.3 [M]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 10.31 (s, 1H), 7.62-7.50 (m, 5H), 6.93 (s, 2H), 5.42-5.39(m, 1H), 4.63-4.20 (m, 2H), 4.05-3.55 (m, 2H), 3.30-3.10 (m, 2H),2.41-2.31 (m, 1H), 2.23 (s, 9H), 2.15-2.06 (m, 2H), 2.05-1.80 (m, 3H),1.57-1.43 (m, 2H), 1.23-1.07 (m, 3H).

Synthesis of 1-benzyl-1-(2-((4-((tert-butoxycarbonyl) (butyl) amino)phenyl) amino)-2-oxoethyl) azepan-1-ium bromide

Synthesis of 2-bromo-N-(4-nitrophenyl) acetamide

To a stirred suspension of 4-nitro aniline (10 g, 72.395 mmol) in H₂O(100 mL) was added bromo acetyl bromide (29.226 g, 144.790 mmol) at 0°C., and the reaction mixture was stirred at room temperature for 16 h asprogress of the reaction was monitored by TLC (50% EtOAc in Pet-ether,Visualization: UV). The reaction mixture was basified with sat.Na₂CO₃solution (100 mL) and the precipitated solid was filtered and dried.Crude product was triturated with diethyl ether (2×100 mL) to afford2-bromo-N-(4-nitrophenyl) acetamide (8 g) as a yellow solid. Mass (ESI):m/z 259.17 [M+1]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.97 (s, 1H), 8.25(d, 2H), 7.83 (d, 2H), 4.11 (s, 2H).

Synthesis of 2-(azepan-1-yl)-N-(4-nitrophenyl) acetamide

To a stirred solution of 2-bromo-N-(4-nitrophenyl) acetamide (5 g,19.300 mmol) in ACN (50 ml) was added K₂CO₃ (8 g, 57.900 mmol) followedby Azepane (3.8 g, 38.600 mmol, 2 eq), and the reaction mixture washeated to 90° C. for 16 h in a sealed tube as progress of the reactionwas monitored by TLC (50% EtOAc in Pet ether, Visualization: UV). Thereaction mixture was allowed to cool to room temperature, and was thendiluted with water (200 mL) and extracted with EtOAc (2×200 mL). Thecombined organic extracts were washed with brine solution (100 mL),dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford crude product. The crude product was triturated withdiethyl ether (2×50 mL) to afford pure 2-(azepan-1-yl)-N-(4-nitrophenyl)acetamide (2.5 g,). Mass (ESI): m/z 278.27 [M+1]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 10.26 (s, 1H), 8.21 (d, 1H), 7.91 (d, 2H), 3.33-3.4 (m,2H), 2.72-2.81 (m, 4H), 1.56-1.62 (m, 8H).

Synthesis of N-(4-aminophenyl)-2-(azepan-1-yl) acetamide

To a stirred solution of 2-(azepan-1-yl)-N-(4-nitrophenyl) acetamide(2.5 g, 9.014 mmol) in MeOH (25 mL) was added 10% Pd/C (2.5 g) at roomtemperature, and the reaction mixture was stirred at RT for 16 h underH₂ gas (Balloon pressure) as progress of the reaction was monitored byTLC (50% EtOAc/pet-ether, Visualization: UV). The reaction mixture wasfiltered, and the filtrate was concentrated under reduced pressure toafford pure N-(4-aminophenyl)-2-(azepan-1-yl) acetamide (1.5 g). Mass(ESI): m/z 248.30 [M+H]. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.20 (s, 1H),7.22 (d, 2H), 6.49 (d, 2H), 4.85 (s, 1H), 3.16-3.19 (m, 2H), 2.68-2.72(m, 4H), 1.57-1.62 (m, 8H).

Synthesis of 2-(azepan-1-yl)-N-(4-(butylamino) phenyl) acetamide

To a stirred solution of N-(4-aminophenyl)-2-(azepan-1-yl) acetamide(1.8 g, 7.277 mmol) and butyronitrile (2.51 g, 36.385 mmol) in methanol(20 mL) was added 10% Pd/C (1.8 g), and the reaction mixture was stirredfor 16 h at room temperature under H₂ gas atmosphere (balloon) asprogress of the reaction was monitored by TLC (50% EtOAc in Pet ether,Visualization: UV).

The reaction mixture was filtered, and the filtrate was concentratedunder reduced pressure to afford desired product2-(azepan-1-yl)-N-(4-(butylamino) phenyl) acetamide (1 g). Mass (ESI):m/z 304.43[M+1]⁺.

Synthesis of tert-butyl (4-(2-(azepan-1-yl) acetamido) phenyl) (butyl)carbamate

To a stirred solution of 2-(azepan-1-yl)-N-(4-(butylamino) phenyl)acetamide (1.4 g, 4.613 mmol) in ACN (20 mL) was added triethyl amine(1.39 g, 13.839 mmol) followed by di-tert-butyl dicarbonate (1.5 g,6.919 mmol) and the reaction mixture was stirred at rt for 16 h asprogress of the reaction was monitored by TLC (50% EtOAc in Pet-ether,Visualization: UV). After completion of starting material on TLC, thecrude compound was diluted with water (100 mL), extracted with EtOAc(2×120 mL) and the combined organic extracts were washed with brinesolution (70 mL), dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. The resulting crude product was purified bycolumn chromatography (eluted with 10%-20% EtOAc in pet-ether) to affordproduct tert-butyl (4-(2-(azepan-1-yl) acetamido) phenyl) (butyl)carbamate (1 g,). Mass (ESI): m/z 404.58 [M+H]. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 9.65 (s, 1H), 7.59 (d, 2H), 7.13 (d, 2H), 3.53 (t, 2H),3.24 (s, 2H), 2.70-2.75 (m, 4H), 1.61-1.86 (m, 8H), 1.35-1.44 (m, 9H),1.24-1.32 (m, 3H), 1.17-1.22 (m, 3H).

Synthesis of 1-benzyl-1-(2-((4-((tert-butoxycarbonyl) (butyl) amino)phenyl) amino)-2-oxoethyl) azepan-1-ium bromide

To a stirred solution of tert-butyl (4-(2-(azepan-1-yl) acetamido)phenyl) (butyl) carbamate (200 mg, 0.495 mmol) in ACN (2 mL) was addedbenzyl bromide (127 mg, 0.742 mmol) at room temperature, and thereaction mixture was stirred at 90° C. for 16 h as progress of thereaction was monitored by TLC (10% MeOH in DCM, Visualization: UV). Thereaction was concentrated under reduced pressure and crude compound wastriturated with diethyl ether (2×20 mL) and EtOAc (2×20 mL) to afford1-benzyl-1-(2-((4-((tert-butoxycarbonyl) (butyl) amino) phenyl)amino)-2-oxoethyl) azepan-1-ium bromide (80 mg). Mass (ESI): m/z 494.56[M]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 10.63 (s, 1H), 7.66 (d, 2H),7.50-7.60 (m, 5H), 7.24 (d, 2H), 4.91 (s, 2H), 4.00 (s, 2H), 3.52-3.76(m, 6H), 1.90-1.98 (m, 4H), 1.63-1.76 (m, 4H), 1.37-1.41 (m, 13H), 0.85(t, 3H).

Synthesis of 1-benzyl-1-(2-((4-(butylamino) phenyl) amino)-2-oxoethyl)azepan-1-ium formate

Synthesis of 1-benzyl-1-(2-((4-(butylamino) phenyl) amino)-2-oxoethyl)azepan-1-ium formate

A reaction mixture of 1-benzyl-1-(2-((4-((tert-butoxycarbonyl) (butyl)amino) phenyl) amino)-2-oxoethyl) azepan-1-ium bromide (50 mg, 0.087mmol) in 47% Aq. HBr (1 mL) was stirred at 80° C. for 16 h as progressof the reaction was monitored by LCMS. The reaction mixture wasconcentrated under reduced pressure to afford crude product which waspurified by reverse phase Prep.HPLC (Column: X-Select C18 (19×250) mm5u, Mobile phase: 0.1% Formic Acid in H₂O: CAN, Flow: 18 ml/min,Gradient method: 0/51, 7.1/50, 7.2/99, 9.2/99, 9.3/5, 12/5). Purefractions were collected and lyophilized to afford1-benzyl-1-(2-((4-(butylamino) phenyl) amino)-2-oxoethyl) azepan-1-iumformate (25 mg). Mass (ESI): m/z 394.53 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆)δ ppm 10.95 (s, 1H), 8.56 (s, 1H), 7.68 (d, 2H), 7.49-7.54 (m, 3H), 7.36(d, 2H), 6.54 (d, 2H), 5.52 (t, 1H), 4.89 (s, 2H), 4.01 (s, 2H),3.67-3.69 (m, 2H), 3.51-3.53 (m, 2H), 2.97 (t, 2H), 1.91-2.01 (m, 4H),1.49-1.62 (m, 2H), 1.35-1.40 (m, 2H), 1.36 (t, 3H).

Synthesis of 1-benzyl-1-(2-((4-(butylamino) phenyl) amino)-2-oxoethyl)piperidin-1-ium carbonate

Synthesis of N-(4-nitrophenyl)-2-(piperidin-1-yl) acetamide

To a stirred solution of 2-bromo-N-(4-nitrophenyl) acetamide (3.0 g,11.580 mmol) in ACN (30.0 mL) was added potassium carbonate (4.801 g,34.74 mmol) and piperidine (1.972 g, 23.16 mmol) and the resultingreaction mixture was stirred at 85° C. for 1 h as progress of thereaction was monitored by TLC (50% ethyl acetate in Pet ether,visualization by UV). The reaction mixture was cooled to rt andconcentrated under reduced pressure to afford crude reside. The cruderesidue was diluted with water (150 mL), extracted with EtOAc (3×100mL), and the combined organic extracts were was washed with brinesolution (1×100 mL), dried over anhydrous sodium sulphate, filtered andconcentrated under reduced pressure to afford crude product which waspurified by column chromatography (eluted with 5% EtOAc in Pet ether) toafford product N-(4-nitrophenyl)-2-(piperidin-1-yl) acetamide (1.40 g)as a pale yellow solid. MS (ESI): m/z 264.26 [M+H⁺]. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 10.28 (s, 1H), 8.20-8.23 (m, 2H), 7.89-7.93 (m, 2H), 3.14(s, 2H), 2.44-2.51 (m, 4H), 1.53-1.59 (m, 4H), 1.40 (t, 2H).

Synthesis of N-(4-aminophenyl)-2-(piperidin-1-yl) acetamide

To a solution of N-(4-nitrophenyl)-2-(piperidin-1-yl) acetamide (1.4 g,5.3171 mmol) in MeOH (15.0 ml) was added 10% Pd/C (1.4 g) and theresulting reaction mixture was stirred at RT for 16 h under hydrogen gasatmosphere (balloon pressure) as progress of the reaction was monitoredby TLC (50% EtOAc in Pet ether, Visualization: UV). The reaction mixturewas filtered through celite washed with an excess of methanol (2×20 ml)and the combined filtrate was concentrated under reduced pressure toafford crude product which was triturated with diethyl ether (3×20 mL)to afford N-(4-aminophenyl)-2-(piperidin-1-yl) acetamide (1.20 g). MS(ESI): m/z 234.29 [M+]. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 10.25 (s, 1H),8.22 (d, 2H), 7.92 (t, 2H), 3.32 (d, 2H), 2.72-2.85 (m, 4H), 1.52-1.62(m, 8H). Analytical data enclosed.

Synthesis of N-(4-(butyl amino) phenyl)-2-(piperidin-1-yl) acetamide

To a solution of N-(4-aminophenyl)-2-(piperidin-1-yl) acetamide (1.2 g,5.1431 mmol) in methanol (15.0 mL) was added butyronitrile (1.777 g,25.7157 mmol) and 10% Pd/C (1.2 g) under nitrogen atmosphere. Theresulting reaction mixture was stirred at rt for 16 h under hydrogen gasatmosphere (balloon pressure) as progress of the reaction was monitoredby TLC (ethyl acetate in Pet ether, UV visualization). The reactionmixture was filtered through celite washed with excess of methanol (2×20mL) and concentrated to afford crude product which was purified bycolumn chromatography (eluted with 20% of EtOAc in Pet ether) to affordpure N-(4-(butyl amino) phenyl)-2-(piperidin-1-yl) acetamide (1.10 g).MS (ESI): m/z 290.42 [M+H]. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.21 (s,1H), 7.27 (d, 2H), 6.48 (d, 2H), 5.33 (t, 2H), 2.88-2.95 (m, 4H),2.49-2.51 (m, 4H), 1.47-1.58 (m, 6H), 1.34-1.41 (m, 4H), 0.90 (t, 3H).

Synthesis of tert-butyl butyl (4-(2-(piperidin-1-yl) acetamido) phenyl)carbamate

To a stirred solution of N-(4-(butylamino) phenyl)-2-(piperidin-1-yl)acetamide (0.956 g, 3.30 mmol) in DCM (10.0 mL) at 0° C. was added TEA(1.672 g, 16.527 mmol) and di-tert-butyl dicarbonate (1.803 g, 8.2635mmol) and the resulting reaction mixture was allowed to stir at rt for16 h as progress of the reaction was monitored by TLC (100% EtOAc,Visualization: UV). The reaction mixture was concentrated under reducedpressure, diluted with water (50 mL) and extracted with DCM (3×50 mL).The combined organic extracts were washed with brine solution (1×50 mL),dried over anhydrous sodium sulphate, filtered and concentrated underreduced pressure to afford crude product which was purified by normalphase flash chromatography (eluted with 30% in Pet ether). The collectedpure fractions were concentrated under reduced pressure to affordproduct tert-butyl butyl (4-(2-(piperidin-1-yl) acetamido) phenyl)carbamate (1.15 g). MS (ESI): m/z 390.57 [M+H]⁺.

Synthesis of 1-benzyl-1-(2-((4-((tert-butoxycarbonyl) (butyl) amino)phenyl) amino)-2-oxoethyl) piperidin-1-ium bromide

To a stirred solution of tert-butyl butyl (4-(2-(piperidin-1-yl)acetamido) phenyl) carbamate (0.225 g, 0.5776 mmol) in Acetonitrile (3mL) was added benzyl bromide (0.197 g, 1.1552 mmol) in microwave vial.The reaction mixture was stirred at 100° C. for 1 h in a microwave (CEMinstrument) as progress of the reaction was monitored by LCMS and TLC(5% MeOH in DCM, Detection: UV). The reaction mixture was allowed tocool to rt and concentrated under reduced pressure to afford crudeproduct which was triturated with diethyl ether (5×5 ml) to affordproduct 1-benzyl-1-(2-((4-((tert-butoxycarbonyl)(butyl)amino)phenyl)amino)-2-oxoethyl) piperidin-1-ium bromide (110 mg). MS (ESI): m/z480.58 [M]+.

Synthesis of 1-benzyl-1-(2-((4-(butyl amino) phenyl) amino)-2-oxoethyl)piperidin-1-ium carbonate

A reaction mixture of 1-benzyl-1-(2-((4-((tert-butoxy carbonyl) (butyl)amino) phenyl) amino)-2-oxoethyl) piperidin-1-ium bromide (105 mg,0.2184 mmol) in aqueous hydrogen bromide (48%) (1 mL) was stirred at rtfor 16 h as progress of the reaction was monitored by LCMS and TLC (80%Ethyl acetate in pet ether, Detection: UV). The reaction mixture waslyophilized to afford crude product which was purified by reverse phasePrep. HPLC (Column: X-select C18 (19*250) 5u, Mobile Phase (A): 10 mMammonium bicarbonate, Mobile Phase (B): 100% ACN Method: 0/3, 2/40,20/40, 20.50/100, 30/35, Flow Rate: 18 ml/min, Solubility:CAN+THF+MeOH). Pure fractions were combined and lyophilized to afford1-benzyl-1-(2-((4-(butyl amino) phenyl) amino)-2-oxoethyl)piperidin-1-ium carbonate (15.1 mg). MS (ESI): m/z 380.2 [M]+. ¹H NMR(400 MHz, DMSO-d₆) δ ppm 11.10 (br s, 1H), 7.54-7.72 (m, 5H), 7.34 (d,2H), 6.52 (d, 2H), 5.44 (t, 1H), 4.94 (s, 2H), 4.09 (s, 2H), 3.65 (d,2H), 3.33-3.5 (m, 2H), 2.97-3.01 (m, 2H), 1.92-1.97 (m, 4H), 1.63 (q,1H), 1.48-1.55 (m, 3H), 1.34-1.42 (m, 2H), 0.91 (t, 3H).

Synthesis of 1-benzyl-1-(1-((4-(butylamino)-2,6-dimethylphenyl)amino)-1-oxobutan-2-yl) piperidin-1-ium carbonate

Synthesis of N-(2,6-dimethylphenyl)-4-methylbenzenesulfonamide

To a stirred solution of 2,6-dimethyl aniline (50 g, 413 mmol) inpyridine (1.2 L) was added Tosyl chloride (94.396 g, 495.131 mmol) andthe mixture was heated to reflux at 115° C. for 4 h as progress of thereaction mixture monitored by TLC (30% Ethyl acetate in pet ether,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude residue which was diluted with water (500 mL),adjusted pH to 6 with 2N HCl (500 mL) and extracted with ethyl acetate(2×1 L). The combined organic extracts were washed with brine solution(1.0 L), dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to afford crude product which was triturated with pet ether toprovide N-(2,6-dimethylphenyl)-4-methylbenzenesulfonamide (90 g) as anoff white solid. MS (ESI): m/z 276.23 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆)δ ppm 9.231 (s, 1H), 7.530-7.551 (d, 2H), 7.354-7.375 (d, 2H),6.985-7.069 (m, 3H), 2.384 (s, 3H), 1.940 (s, 6H).

Synthesis of N-(2,6-dimethyl-4-nitrophenyl)-4-methylbenzenesulfonamide

To a solution of N-(2,6-dimethylphenyl)-4-methylbenzenesulfonamide (90g, 326.8 mmol) in AcOH (675 mL) and water (450 mL) was added NaNO₂(45.103 g, 653.666 mmol) followed by th drop-wise addition of Conc.HNO₃(41.181 g, 653.666 mmol) over a period of 15 min at room temperature.The resulting reaction mixture was heated to 110° C. for 5 h as progressof the reaction mixture was monitored by TLC (30% ethyl acetate in petether, Visualization: UV). The reaction mixture was diluted with icecold water (500 mL), basified with 1N NaOH solution (800 mL) andextracted with ethyl acetate (2×1 L). The combined organic extracts werewas washed with brine solution (1 L), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford crude product which wastriturated with pet ether (500 mL) to provide productN-(2,6-dimethyl-4-nitrophenyl)-4-methylbenzenesulfonamide (40 g) as anoff white solid. MS (ESI): m/z 321.18 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆)δ ppm 9.72 (s, 1H), 7.925 (s, 2H), 7.562-7.583 (d, 2H), 7.390-7.409 (d,2H), 2.400 (s, 3H), 2.073 (s, 6H).

Synthesis of 2,6-dimethyl-4-nitroaniline

To a stirred solution ofN-(2,6-dimethyl-4-nitrophenyl)-4-methylbenzenesulfonamide (25 g, 78.037mmol) in AcOH (125 ml) was added HClO₄ (250 ml) at rt and the mixturewas heated to 100° C. for 3 h. as progress of the reaction mixture wasmonitored by TLC (30% Ethyl acetate in pet ether, Visualization: UV).The reaction mixture was poured into crushed ice, basified (pH ˜11) withaqueous ammonia solution and extracted with ethyl acetate (2×1 L). Thecombined organic extracts were washed with brine solution (300 mL),dried over anhydrous Na₂SO₄ and concentrated under reduced pressure toafford crude product which was purified by column chromatography (elutedwith 20% ethyl acetate in pet ether) to afford2,6-dimethyl-4-nitroaniline (10 g) as a yellow solid. MS (ESI): m/z167.03 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.787 (s, 2H), 6.142 (s,2H), 2.155 (s, 6H).

Synthesis of 2-bromo-N-(2,6-dimethyl-4-nitrophenyl) butanamide

To a cooled solution (0° C.) of 2,6-dimethyl-4-nitroaniline (3 g, 18.05mmol) in DCM (45 mL) was added pyridine (1.713 g, 21.66 mmol) followedby 2-bromobutanoyl chloride (4.017 g, 21.66 mmol). The resultingreaction mixture was allowed to stir at rt for 16 h as progress of thereaction mixture was monitored by TLC (50% ethyl acetate in pet ether,Visualization: UV). The reaction mixture was filtered and the isolatedsolid was washed with DCM (20 mL) and dried under high vacuum to afford2-bromo-N-(2,6-dimethyl-4-nitrophenyl) butanamide (2 g) as an off whitesolid. MS (ESI): m/z 315.26 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.05(s, 1H), 8.0 (s, 2H), 4.55 (t, 1H), 2.279 (s, 6H), 1.940-2.143 (m, 2H),0.99 (t, 3H).

Synthesis of N-(2,6-dimethyl-4-nitrophenyl)-2-(piperidin-1-yl)butanamide

To a stirred solution of 2-bromo-N-(2,6-dimethyl-4-nitrophenyl)butanamide (2 g, 6.345 mmol) in ACN (30 mL) was added K₂CO₃ (2.627 g,19.03 mmol) and piperidine (1.080 g, 12.690 mmol). The resulting mixturewas heated to 80° C. for 16 h as progress of the reaction mixture wasmonitored by TLC (50% ethyl acetate in pet ether, Visualization: UV).The reaction mixture was concentrated, diluted with Ethyl acetate (150mL) and washed with water (50 mL×3) and brine solution (60 mL). Thecombined organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford crude product which waspurified by column chromatography (eluted with 50% ethyl acetate in petether) to afford N-(2,6-dimethyl-4-nitrophenyl)-2-(piperidin-1-yl)butanamide (1.70 g). MS (ESI): m/z 320.42 [M+H]⁺. ¹H NMR (500 MHz,DMSO-d₆) δ ppm 9.578 (s, 1H), 7.992 (s, 2H), 3.12 (t, 1H), 2.55-2.62 (m,4H), 2.28 (s, 6H), 1.60-1.81 (m, 2H), 1.35-1.56 (m, 6H), 0.93 (t, 3H).

Synthesis of N-(4-amino-2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide

To a stirred solution ofN-(2,6-dimethyl-4-nitrophenyl)-2-(piperidin-1-yl) butanamide (1.7 g,5.322 mmol) in EtOH (40 mL) was added 10% Pd—C(0.900 g) and the mixturewas stirred for 16 h at rt under H₂ gas (balloon pressure) as progressof the reaction mixture was monitored by TLC (100% Ethyl acetate,Visualization: UV). The mixture was filtered through celite, the filterbed was washed with MeOH (200 ml) and the filtrate was concentratedunder reduced pressure to afford crude product which was purified bynormal phase flash chromatography (eluted with EtOAc) to affordN-(4-amino-2,6-dimethylphenyl)-2-(piperidin-1-yl) butanamide (1.0 g). MS(ESI): m/z 290.46 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.78 (s, 1H),6.23 (s, 2H), 4.82 (s, 2H), 2.95 (t, 1H), 2.45-2.6 (m, 4H), 1.2-1.6 (m,8H), 0.910 (t, 3H).

Synthesis of N-(4-(butylamino)-2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide

To a solution of N-(4-amino-2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide (1.0 g, 3.455 mmol) in MeOH (20 mL) was added butyronitrile(2.387 g, 34.551 mmol) and 10% Pd/C (1 g) at and the mixture was stirredfor 16 h at rt under H₂ gas (balloon pressure) as progress of thereaction mixture was monitored by TLC (Ethyl acetate, Visualization:UV). The reaction mixture was filtered through a bed of celite, thefilter bed was washed with MeOH (150 ml) and the filtrate wasconcentrated under reduced pressure to afford crude product which waspurified by normal phase flash chromatography (eluted with ethylacetate) to afford N-(4-(butylamino)-2,6-dimethylphenyl)-2-(piperidin-1-yl) butanamide (0.500 g). MS (ESI): m/z 346.51[M+H]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 8.789 (s, 1H), 6.242 (s, 2H),2.96 (t, 3H), 2.50-2.57 (m, 3H), 2.027 (s, 6H), 1.498-1.518 (m, 2H),1.475-1.488 (m, 6H), 1.22-1.387 (m, 6H), 0.885-0.918 (m, 6H).

Synthesis of tert-butyl butyl (3,5-dimethyl-4-(2-(piperidin-1-yl)butanamido) phenyl) carbamate

To a stirred solution ofN-(4-(butylamino)-2,6-dimethylphenyl)-2-(piperidin-1-yl) butanamide(0.400 g, 1.157 mmol) in EtOH (10 mL) was added DIPEA (0.179 g, 1.389mmol) and di-tert-butyl dicarbonate (0.758 g, 3.473 mmol) at 0° C. Thereaction mixture was stirred at rt for 16 h as progress of the reactionmixture was monitored by TLC (50% Ethyl acetate in pet ether,Visualization: UV). The mixture was concentrated under vacuum to affordcrude reside which was diluted with ethyl acetate (80 ml) and washedwith water (40 ml), brine solution (40 ml). The organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to affordcrude product which was purified by normal phase flash chromatography(eluted with 40% ethyl acetate in pet ether) to afford tert-butyl butyl(3,5-dimethyl-4-(2-(piperidin-1-yl) butanamido) phenyl) carbamate (0.300g). MS (ESI): m/z 446.66 [M+H]⁺. ¹H NMR data shows product along withaliphatic impurities. Analytical data enclosed.

Synthesis of 1-benzyl-1-(1-((4-((tert-butoxycarbonyl) (butyl)amino)-2,6-dimethylphenyl) amino)-1-oxobutan-2-yl) piperidin-1-iumbromide

To a stirred solution of tert-butyl butyl(3,5-dimethyl-4-(2-(piperidin-1-yl) butanamido) phenyl) carbamate (0.250g, 0.561 mmol) in ACN (5 mL) was added benzyl bromide (0.191 g, 1.116mmol) and the mixture was heated to 85° C. for 24 h as progress of thereaction mixture was monitored by TLC (10% Methanol in DCM,Visualization: UV). The reaction mixture was concentrated under vacuumto afford crude product which was triturated with mixture of diethylether (20 ml) and ethyl acetate (10 ml) to afford1-benzyl-1-(1-((4-((tert-butoxycarbonyl) (butyl)amino)-2,6-dimethylphenyl) amino)-1-oxobutan-2-yl)piperidin-1-iumbromide (0.200 g). MS (ESI): m/z 536.61 [M]⁺. ¹H NMR (500 MHz, DMSO-d₆)δ ppm 10.317 (s, 1H), 7.547 (s, 5H), 7.002 (s, 2H), 5.41 (d, 1H), 4.62(d, 1H), 3.6-3.9 (m, 3H), 3.1-3.4 (m, 2H), 2.4-2.5 (m, 2H), 1.953-2.184(m, 12H), 1.163-1.620 (m, 18H), 0.857 (s, 3H).

Synthesis of 1-benzyl-1-(1-((4-(butylamino)-2,6-dimethylphenyl)amino)-1-oxobutan-2-yl) piperidin-1-ium carbonate

An Aqueous 47% HBr solution (8 ml) was added to1-benzyl-1-(1-((4-((tert-butoxycarbonyl)(butyl)amino)-2,6-dimethylphenyl)amino)-1-oxobutan-2-yl) piperidin-1-iumbromide (0.150 g, 0.243 mmol) at 0° C. and the resulting mixture wasallowed to stir at rt for 16 h as progress of the reaction mixture wasmonitored by TLC. (10% Methanol in DCM, Visualization: UV). The reactionmixture was concentrated under reduced pressure and the residue waswashed with diethyl ether (5 ml) to afford crude product which waspurified by reverse phase prep-HPLC (Column: kromasil C18 (25×150) mm10u, Mobile phase-10 mM Ammonium Bicarbonate in H₂O: CAN, Flow: 25ml/min, Gradient method: 0/40, 10/82, 10.1/99, 12/99, 12.1/40, 14/40).The pure fractions were collected and lyophilized to afford1-benzyl-1-(1-((4-(butyl amino)-2,6-dimethylphenyl)amino)-1-oxobutan-2-yl) piperidin-1-ium carbonate salt (0.050 g). MS(ESI): m/z 436.61 [M]⁺. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 10.826 (s, 1H),7.54 (t, 5H), 6.271 (s, 2H), 5.44 (t, 2H), 4.60 (d, 2H), 3.708 (s, 2H),2.96 (q, 2H), 2.362 (s, 2H). 2.027-2.066 (m, 8H), 1.838-1.99 (m, 2H),1.492-1.549 (m, 6H), 1.32-1.45 (m, 3H), 1.12-1.25 (m, 3H), 0.922 (t,3H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(methoxy carbonyl) piperidin-1-ium bromide

Synthesis of methyl 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-4-carboxylate

To a stirred solution of 2-bromo-N-(2,6-dimethylphenyl) acetamide (0.500g, 2.065 mmol) and methyl piperidine-4-carboxylate (0.443 g, 3.097 mmol)in ACN (10 ml) was added K₂CO₃ (0.712 g, 5.162 mmol). The mixture washeated to 90° C. for 16 h as progress of the reaction mixture wasmonitored by TLC (50% Ethyl acetate in pet ether, Visualization: UV).The reaction mixture was concentrated under reduced pressure, dilutedEtOAc (100 ml) and washed with water (50 ml×3) and brine solution (50ml). The organic extract was dried over anhydrous Na₂SO₄ andconcentrated to afford crude product which was purified by normal phaseflash chromatography (eluted with 10%-30% of Ethyl acetate in Pet ethergradient). The collected pure fractions were concentrated under reducedpressure to afford the desired product methyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) piperidine-4-carboxylate (0.400 g). MS (ESI): m/z305.36 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.171 (s, 1H), 7.060-7.09(m, 3H), 3.607 (s, 3H), 3.089 (s, 2H), 2.879-2.908 (m, 2H), 2.316-2.371(m, 1H), 2.198-2.261 (m, 2H), 2.129 (s, 6H), 1.816-1.856 (m, 2H).1.680-1.778 (m, 2H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(methoxy carbonyl) piperidin-1-ium bromide

To a stirred solution of methyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) piperidine-4-carboxylate (0.200 g, 0.657 mmol) in ACN(5 ml) was added benzyl bromide (0.224 g, 1.314 mmol) and the mixturewas heated to 90° C. for 24 h as progress of the reaction was monitoredby TLC (10% MeOH in DCM, Visualization: UV). The reaction mixture wasconcentrated to afford crude product which was triturated ethyl acetate(30 ml) and n-pentane (20 ml) to afford pure1-benzyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)-4-(methoxycarbonyl) piperidin-1-ium bromide (0.080 g) as white solid. MS (ESI):m/z 395.3 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.048 (s, 1H),7.551-7.591 (m, 5H), 7.144-7.159 (m, 3H), 4.947 (s, 2H), 4.237 (s, 2H),3.671-3.766 (m, 5H), 3.541-3.549 (m, 2H), 2.732-2.788 (m, 1H),2.128-2.212 (m, 10H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethoxycarbonyl) piperidin-1-ium bromide

Synthesis of ethyl 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxylate

To a stirred solution of 2-chloro-N-(2,6-dimethylphenyl) acetamide (5 g,25.295 mmol) in ACN (100 ml) was added K₂CO₃ (8.726 g, 63.237 mmol) andethyl piperidine-3-carboxylate (5.964 g, 37.942 mmol) at RT. Theresulting reaction mixture was stirred at RT for 16 h as progress of thereaction mixture was monitored by TLC (30% Ethyl acetate in pet ether,Visualization: UV). The reaction mixture was filtered to removeinorganic salts and the filtrate was concentrated under reduced pressureto afford crude product which was diluted with ethyl acetate (250 ml)and washed with water (80 ml×3) and brine solution (80 ml). The organicphase was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to afford ethyl 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxylate (6.5 g). MS (ESI): m/z 319.01 [M+H]⁺. ¹H NMR(400 MHz, CDCl₃) δ ppm 8.751 (s, 1H), 7.066-7.101 (m, 3H), 4.075-4.168(m, 2H), 3.154-3.249 (m, 2H), 2.927-2.949 (m, 1H), 2.761-2.817 (m, 2H),2.613-2.664 (m, 1H), 2.45-2.49 (m, 1H), 2.23 (s, 6H), 1.506-2.045 (m,4H), 1.209-1.277 (m, 3H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethoxycarbonyl) piperidin-1-ium bromide

To a stirred solution of ethyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) piperidine-3-carboxylate (0.300 g, 0.942 mmol) in ACN(10 ml) was added benzyl bromide (0.322 g, 1.884 mmol) and the mixturewas heated to 90° C. for 16 h in sealed tube as progress of the reactionwas monitored by TLC (10% MeOH in DCM, Visualization: UV). The reactionmixture was concentrated under reduced pressure to afford crude productwhich was purified by normal phase flash chromatography (eluted with 2%Methanol in DCM). The collected pure fractions were concentrated underreduced pressure to afford the desired product as a colourless gum(LCMS-85%), This compound was diluted with ethyl acetate (15 ml) andstirred for 1 h, and then filtered and washed with ethyl acetate (15ml×3) to afford pure 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethoxycarbonyl) piperidin-1-ium bromide (70 mg) asan off white solid. MS (ESI): m/z 409.2 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆)δ ppm 10.05 (s, 1H), 7.545-7.585 (m, 5H), 7.125-7.178 (m, 3H),4.945-5.060 (m, 2H), 4.109-4.342 (m, 4H), 3.904-3.934 (m, 1H),3.644-3.677 (m, 1H), 3.433-3.569 (m, 2H), 3.230-3.262 (m, 1H), 2.213 (s,6H), 1.948-2.195 (m, 3H), 1.553-1.620 (m, 1H), 1.24 (t, 3H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(isopropoxycarbonyl) piperidin-1-iumtrifluoroacetate salt

Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylic acid

A mixture of ethyl 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxylate (5 g, 15.702 mmol) in Conc. HCl (100 ml) washeated to 100° C. for 16 h as progress of the reaction was monitored byTLC (50% EtOAc in pet ether, Visualization: UV). The reaction mixturewas concentrated under reduced pressure to afford1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl) piperidine-3-carboxylicacid (4.2 g) MS (ESI): m/z 291.13 [M+H]⁺.

Synthesis of isopropyl 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxylate

To a stirred solution of 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxylic acid (0.5 g, 1.722 mmol) in isopropanol (10 mL)was added thionyl chloride (5 mL) drop wise at 0° C. The resultingreaction mixture was allowed to stir at 90° C. for 16 h as progress ofthe reaction was monitored by TLC (50% EtOAc in pet ether,Visualization: UV). The reaction mixture was cooled to room temperatureand concentrated under reduced pressure to afford crude residue whichwas quenched with ice cold saturated sodium bicarbonate solution (20 mL)and extracted with EtOAc (2×50 mL). The combined organic extracts weredried over anhydrous sodium sulphate and concentrated under reducedpressure to afford crude product which was purified by normal phaseflash chromatography (eluted with 20%-30% of EtOAc/Pet ether). Thecollected pure fractions were concentrated under reduced pressure toafford 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxylate (0.54 g) as a pale yellow solid. MS (ESI): m/z332.27 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.5 (br s, 1H), 7.06-7.11(m, 3H), 4.97-5.03 (m, 1H), 3.19-3.24 (m, 2H), 2.93-2.95 (m, 1H),2.59-2.48 (m, 2H), 2.48-2.40 (m, 2H), 2.18-2.20 (m, 5H), 1.41-1.65 (m,3H), 1.18-1.24 (m, 6H).

Synthesis of1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(isopropoxycarbonyl) piperidin-1-ium trifluoroacetate salt

To a stirred solution of isopropyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) piperidine-3-carboxylate (0.4 g, 1.203 mmol) in ACN(10 mL) was added benzyl bromide (0.6173 g, 3.609 mmol) at roomtemperature. The resulting reaction mixture was stirred at 90° C. for 16h in a sealed tube as progress of the reaction was monitored by TLC (50%EtOAc in Pet ether, Visualization: UV). The reaction mixture was allowedto cool to room temperature and was concentrated under reduced pressureto afford 430 mg (LCMS 51%) of crude compound which was further purifiedby reverse phase Prep.HPLC (Column: X-select csh C18 (250*19) mm, 5u;Mobile Phase A: 0.1% TFA (Aq.) in Water, Mobile Phase B: Acetonitrile,Flow: 22 ml/min, Method (T/% of B): 0/10, 2/10, 10/50, 13/50, 13.1/100,16/100, 16.1/10, 19/20, Solubility: ACN+H₂O+THF, Temperature: Ambient).Combined pure fractions were lyophilized to afford pure1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(isopropoxycarbonyl) piperidin-1-iumtrifluoroacetate salt (168 mg). LCMS: 99.37% (77.95%+21.42%, mixture ofisomers), MS (ESI): m/z 423.2, [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm10.01-10.05 (m, 1H), 7.57-7.61 (m, 5H), 7.12-7.17 (m, 3H), 4.92-5.06 (m,3H), 4.14-4.22 (m, 2H), 3.87-3.67 (dd, 1H), 3.46-3.55 (m, 3H), 3.15-3.21(m, 1H), 2.208 (d, 6H), 1.85-2.07 (m, 3H), 1.19-1.23 (m, 6H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propoxycarbonyl) piperidin-1-ium bromide

Synthesis of propyl piperidine-3-carboxylate

To a stirred solution of piperidine-3-carboxylic acid (2.0 g, 15.484mmol) in propan-1-ol (20.0 ml) was added SOCl₂ (11.05 g, 92.908 mmol) at0-5° C. The resulting reaction mixture was stirred for 16 h at 80° C. asprogress of the reaction was monitored by TLC (30% ethyl acetate/petether. Visualisation: ninhydrin). The reaction mixture was concentratedunder reduced pressure to afford the crude compound which was dissolvedinto ethyl acetate (100 ml) and wash with saturated bicarbonate solution(1×100 ml) and brine solution (1×50 ml). The organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to affordthe propyl piperidine-3-carboxylate (2.0 g) as a colourless liquid. Thiscrude product carry forwarded to next step without purification andanalysis.

Synthesis of propyl-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxylate

To a stirred solution of 2-chloro-N-(2,6-dimethylphenyl) acetamide (2.0g, 10.15 mmol) in ACN (20 ml) was added K₂CO₃ (4.14 g, 29.95 mmol) andpropyl piperidine-3-carboxylate (3.47 g, 20.28 mmol) at roomtemperature. The resulting reaction mixture was stirred for 16 h at 80°C. as progress of the reaction was monitored by TLC (50% ethylacetate/pet ether, Visualization: UV active). After completion ofreaction on TLC, the reaction mixture was concentrated under reducedpressure to afford the crude compound which was diluted with water (30ml) and extracted with ethyl acetate (2×100 ml). The combined organicextracts were washed with brine (50.0 ml), dried over anhydrous Na₂SO₄and concentrated under reduced pressure to afford the crude compoundwhich was purified by column chromatography (eluted with 20%-80% ofethyl acetate/pet ether). The collected pure fractions were concentratedunder reduced pressure to afford the propyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) piperidine-3-carboxylate (1.0 g) as a gummy compound.MS (ESI): m/z 333.10 [M+H]⁺.

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propoxy carbonyl) piperidin-1-ium bromide

To a solution of propyl 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxylate (0.3 g, 0.9030 mmol) in Acetonitrile (10 ml)was added benzyl bromide (0.3113 g, 1.806 mmol) at room temperature. Theresulting reaction mixture was stirred at 90° C. for 16 h as progress ofthe reaction was monitored by TLC (10% MeOH/DCM, Visualization: UV).After consumption of the starting material on TLC, the reaction mixturewas directly concentrated under reduced pressure to afford crudecompound as pale yellow semi solid which was purified by normal phaseflash chromatography (eluted with 0%-30% of MeOH/DCM). The collectedpure fractions were concentrated under reduced pressure to afford1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propoxycarbonyl) piperidin-1-ium bromide (70 mg)as an off-white solid. MS (ESI): m/z 423.3 [M]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 10.30 (s, 1H), 7.55-7.60 (m, 5H), 7.11-7.18 (m, 3H),4.97-5.15 (m, 2H), 4.33 (dd, 2H) 4.05 (t, 2H), 3.88 (d, 1H), 3.66 (d,1H), 3.31-3.54 (m, 3H), 2.09-2.21 (m, 8H), 1.96 (d, 2H), 1.55-1.64 (m,3H), 0.87-0.93 (m, 3H).

The following examples were prepared from piperidine-3-carboxylic acid,2-chloro-N-(2,6-dimethylphenyl) acetamide and benzyl bromide followingprocedures described for the synthesis of compound 47A.

Compound # Structure MS (ESI): m/z 48A

437.2 [M]⁺ 49A

437.3 [M]⁺ 50A

449.2 [M]⁺ 51A

463.3 [M]⁺.

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propionyloxy) piperidin-1-ium bromide

Synthesis of tert-butyl 3-(propionyloxy) piperidine-1-carboxylate

To a stirred solution of tert-butyl 3-hydroxypiperidine-1-carboxylate (2g, 9.936 mmol) and pyridine (2.357 g, 29.808 mmol) in DCM (30 ml) wasadded propionyl chloride (1.103 g, 11.923 mmol) at 0° C. and theresulting reaction mixture was stirred for 24 h at room temperature asprogress of the reaction was monitored by TLC (20% EtOAc in Pet ether,Visualization: UV). The reaction mass was diluted with DCM (120 mL) andwashed twice with water (50 mL), dried over sodium sulphate andconcentrated under reduced pressure to afford tert-butyl3-(propionyloxy) piperidine-1-carboxylate (1.34 g) as an off-whitesolid. ¹H NMR (400 MHz, CDCl₃) δ ppm 4.85-4.75 (m, 1H), 3.51-3.43 (m,3H), 3.3-3.29 (m, 1H), 2.41-2.39 (m, 2H), 1.85-1.72 (m, 3H), 1.45 (s,9H), 1.18-1.12 (m, 4H).

Synthesis of piperidin-3-yl propionate

To a stirred solution of tert-butyl 3-(propionyloxy)piperidine-1-carboxylate (1.3 g, 5.05 mmol) in DCM (20 ml) was added TFA(5 ml) at 0° C. and the resulting reaction mixture was stirred at roomtemperature for 16 h as progress of the reaction was monitored by TLC(50% EtOAc in Pet ether, Visualization: ninhydrin). The reaction mixturewas concentrated under reduced pressure to afford crude product whichwas co-distilled with toluene (2×15 mL) to afford piperidin-3-ylpropionate TFA salt (1.34 g) as a light yellow oil. ¹H NMR (400 MHz,CDCl₃) δ ppm 5.16 (s, 1H), 3.41 (d, 2H), 3.24 (t, 1H), 3.10 (d, 1H),2.42-2.36 (m, 2H), 2.13-2.10 (m, 2H), 2.02-2.01 (m, 2H), 1.88-1.84 (m,3H).

Synthesis of 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidin-3-yl propionate

To a stirred solution of 2-chloro-N-(2,6-dimethylphenyl)acetamide (0.3g, 1.517 mmol) in Acetonitrile (15 mL) was added DIPEA (0.5884 g, 4.551mmol) and piperidin-3-yl propionate TFA salt (0.4937 g, 1.8204 mmol) atroom temperature. The resulting reaction mixture was stirred at 80° C.for 16 h in a sealed tube as progress of the reaction was monitored byTLC (50% EtOAc in pet ether. Visualization: UV). The reaction mixturewas concentrated under reduced pressure to afford residue which wasdiluted with water (50 ml) and extracted with ethyl acetate (2×25 ml).The combined organic extracts were dried over sodium sulphate andconcentrated under reduced pressure to afford crude product which waspurified by normal phase flash chromatography (eluted with 25%-30% ofEtOAc/Pet ether). The collected pure fractions were concentrated underreduced pressure to afford2-(azepan-1-yl)-N-(3-methyl-[1,1′-biphenyl]-2-yl) acetamide (310 mg).Mass (ESI): 319.26 m/z, [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.67 (s,1H), 7.12-7.06 (m, 3H), 4.95-4.92 (m, 1H), 3.20 (s, 2H), 2.95-2.89 (m,1H), 2.73-2.56 (m, 1H), 2.59-2.54 (m, 2H), 2.31-2.23 (m, 8H), 1.88-1.84(m, 2H), 1.68-1.56 (m, 3H), 1.09 (t, 3H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propionyloxy) piperdin-1-ium bromide

To a stirred solution1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidin-3-yl propionate(0.2 g, 0.6281 mmol) in Acetonitrile (15 ml) was added Benzyl bromide(0.4297 g, 2.5124 mmol) in a sealed tube at room temperature and theresulting reaction mixture was stirred at room temperature for 16 h asprogress of the reaction was monitored by TLC (10% Methanol in DCM.Visualization: UV). The reaction mixture was allowed to cool to roomtemperature and was concentrated under reduced pressure to afford crudeproduct, which was triturated with 1:1 EtOAc:Et₂O (30:30 ml) to afford1-benzyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)-3-(propionyloxy)piperidin-1-ium bromide (90 mg) (mixture of isomers, Peak-1:35.75%+Peak-2: 63.34%). Mass (ESI): m/z 409.03 [M]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 10.05 (d, 1H), 7.60-7.56 (m, 5H), 7.15-7.13 (m, 3H),5.45-5.25 (m, 1H), 5.05-4.97 (m, 1H), 4.35-4.18 (m, 2H), 4.29 (d, 2H),3.87-3.52 (m, 4H), 2.44-2.35 (m, 2H), 2.21 (m, 6H), 2.20-1.98 (m, 3H),1.80-1.6 (m, 1H), 1.04 (t, 3H).

Synthesis of 1-benzyl-3-carbamoyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) piperidin-1-ium bromide

Synthesis of 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxamide

To a stirred solution of 2-chloro-N-(2,6-dimethylphenyl) acetamide (2.0g, 10.1183 mmol) in ACN (20.0 ml) was added potassium carbonate (4.195g, 30.3551 mmol) and the resulting mixture was stirred for 10 min at RT.The reaction mixture was treated with piperidine-3-carboxamide (2.6081g, 20.2366 mmol) at room temperature, and then heated at 90° C. whilestirring for 16 h as progress of the reaction was monitored by TLC (10%MeOH in DCM. Visualization: UV). The reaction mixture was allowed tocool to room temperature, diluted with 70 ml of ethyl acetate and washedwith 2×50 ml of water followed by 1×30 ml of brine solution. The organicphase was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to afford 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxamide (2.5 g) as an off white solid. MS (ESI): m/z290.25 [M+H]⁺. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.57 (s, 1H),7.06-7.09 (m, 3H), 5.95 (br s, 1H), 5.45 (br s, 1H), 3.15-3.30 (m, 2H),2.70-2.90 (m, 3H), 2.45-2.60 (m, 2H), 2.15-2.30 (m, 6H), 1.81-1.86 (m,2H), 1.60-1.72 (m, 2H).

Synthesis of 1-benzyl-3-carbamoyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl) piperidin-1-ium bromide

To a stirred solution of 1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidine-3-carboxamide (0.250 g, 0.865 mmol) in ACN (3.0 ml) was addedbenzyl bromide (0.295 g, 1.730 mmol) and the resulting reaction mixturewas heated at 90° C. for 16 h as progress of the reaction was monitoredby TLC (10% MeOH in DCM, Visualization: UV). The reaction mixture wasdirectly concentrated under reduced pressure to afford crude compoundwhich was triturated with 3×10 ml of ethyl acetate to afford1-benzyl-3-carbamoyl-1-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)piperidin-1-ium bromide (130 mg) as an off white solid (33.18%+65.55%,mixture of isomers). MS (ESI): m/z 380.2 [M]⁺. ¹H NMR (400 MH-DMSO-d6) δppm 9.92-10.15 (m, 1H), 7.45-7.60 (m, 6H), 7.16-7.22 (m, 4H), 4.94-5.06(m, 2H), 4.05-4.29 (m, 2H), 3.41-3.83 (m, 4H), 2.92 (t, 1H), 2.19-2.21(m, 6H), 1.98-2.08 (m, 3H), 1.50-1.54 (m, 1H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethyl carbamoyl) piperidin-1-ium bromide

Synthesis of 3-(ethylcarbamoyl) piperidine-1-carboxylate

To a stirred solution of 1-(tert-butoxycarbonyl) piperidine-3-carboxylicacid (2 g, 8.722 mmol) in THE (20 mL) was added Et₃N (2.64 g, 26.166mmol) and ethanamine hydrochloride (1.42 g, 17.444 mmol) followed byHATU (4.97 g, 13.083 mmol) and the reaction mixture was stirred at roomtemperature for 16 h as progress of the reaction was monitored by TLC(50% EtOAc-Pet-ether, Visualization: Ninhydrine). Upon completion, thereaction mixture was concentrated under reduced pressure, quenched with2N HCl and extracted with EtOAc (2×200 mL).

The combined organic extracts were washed with brine (200 mL), driedover anhydrous Na₂SO₄, filtered, and concentrated under reducedpressured to afford crude compound (2.2 g) which was purified by columnchromatography (eluted with 40% of EtOAc in pet-ether) to afford puretert-butyl 3-(ethylcarbamoyl) piperidine-1-carboxylate (2.1 g) as alight brown liquid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 6.12 (br s, 1H),3.83 (br s, 2H), 2.80 (S, 5H), 2.26-2.28 (m, 1H), 2.04-1.05 (m, 2H),1.84-1.85 (m, 1H), 1.46-1.48 (m, 11H), 1.13 (t, 3H).

Synthesis of N-ethylpiperidine-3-carboxamide

To a stirred solution of tert-butyl 3-(ethylcarbamoyl)piperidine-1-carboxylate (2.5 g, 9.752 mmol) in DCM (20 mL) was addedTFA (10 mL) at 0° C., and the reaction mixture was stirred at roomtemperature for 3 h as progress of the reaction was monitored by TLC(10% MeOH-DCM, Visualization: Ninhydrine). The reaction mixture wasconcentrated under reduced pressure to afford pureN-ethylpiperidine-3-carboxamide TFA salt (2.4 g) as a light brownliquid. LCMS purity: 99.89%, Mass (ESI): m/z 157.09 [M+H]⁺.

Synthesis 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N-ethylpiperidine-3-carboxamide

To a solution of N-ethylpiperidine-3-carboxamide TFA salt (1 g, 3.96mmol) in ACN (10 mL) was added DIPEA (1.53 g, 11.89 mmol) followed by2-bromo-N-(2,6-dimethylphenyl) acetamide (960 mg, 3.96 mmol) and thereaction mixture was stirred at 90° C. for 16 h in a sealed tube asprogress of the reaction was monitored by TLC (10% MeOH-DCM,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude compound (1.5 g) which was purified by columnchromatography (eluted with 2% of MeOH in DCM) to afford product1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N-ethylpiperidine-3-carboxamide (1 g,) as a semisolid. LCMS purity: 39%, Mass (ESI): m/z 318.12 [M+H]⁺. This productcarry forwarded to next step without further purification.

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethylcarbamoyl) piperidin-1-ium bromide

To a stirred solution of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N-ethylpiperidine-3-carboxamide (0.3 g, 0.945 mmol)in acetonitrile (2 ml) was added benzyl bromide (0.808 g, 4.724 mmol)and the resulting reaction mixture was stirred at 90° C. for 16 h asprogress of the reaction was monitored by TLC (10% Methanol in DCM.Visualization: UV). The reaction mixture was allowed to cool to roomtemperature and was concentrated under reduced pressure to afford crudeproduct which was purified by normal phase flash chromatography (elutedwith 10%-15% of MeOH in DCM) to afford1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethylcarbamoyl) piperidin-1-ium bromide (100 mg)as a white solid. MS (ESI): m/z 408.19 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δppm 10.15 (s, 1H), 7.98 (t, 1H), 7.60-7.53 (m, 5H), 7.18-7.12 (m, 3H),5.01-5.00 (m, 2H), 4.22 (s, 2H), 3.73-3.66 (m, 2H), 3.51-3.42 (m, 2H),3.13-3.04 (m, 2H), 2.9-2.78 (m, 1H), 2.21 (s, 6H), 2.02-1.98 (m, 3H),1.54-1.51 (m, 1H), 0.99 (t, 3H).

The following examples were prepared from 1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid, 2-bromo-N-(2,6-dimethylphenyl) acetamideand benzyl bromide following procedures described for the synthesis ofcompound 54A. Products were purified by normal phase flashchromatography or reverse phase prep. HPLC.

Compound # Structure MS (ESI): m/z 55A

394.35 [M]⁺ 56A

422.2 [M]⁺ 57A

422.2 [M]⁺ 58A

436.24 [M]⁺ 59A

436.3 [M]⁺ 60A

448.3 [M]⁺ 61A

462.3 [M]⁺ 62A

434.3 [M]⁺ 63A

448.3 [M]⁺ 64A

462.3 [M]⁺ 65A

408.2 [M]⁺ 66A

436.3 [M]⁺. 67A

464.3 [M]⁺

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(propoxycarbonyl)-1,4-diazepan-1-ium bromide

Synthesis of 1-(tert-butyl) 4-propyl 1,4-diazepane-1,4-dicarboxylate

To a stirred solution of tert-butyl 1,4-diazepane-1-carboxylate (0.5 g,2.496 mmol) in THE (5 ml) was added carbonyl diimidazole (1.21 g, 7.488mmol, 3.0 eq) and TEA (0.758 g, 7.488 mmol) followed by 1-propanol(0.449 g, 7.488 mmol) at 0° C. The resulting reaction mixture wasstirred at 80° C. for 16 h as progress of the reaction was monitored byTLC (30% EtOAc-Hexane, Visualization: PMA,). The reaction mixture wasconcentrated under reduced pressure to afford crude product which wasdiluted with EtOAc (150 ml) and washed with water (3×30 ml). The organicphase was dried over Na₂SO₄ and concentrated under reduced pressure toafford 1-(tert-butyl) 4-propyl 1,4-diazepane-1,4-dicarboxylate (0.655g). MS (ESI): m/z 287.19 [M+H]⁺.

Synthesis of propyl 1,4-diazepane-1-carboxylate

A solution of 4M Dioxane.HCl (10 ml) was added to 1-(tert-butyl)4-propyl 1,4-diazepane-1,4-dicarboxylate (0.65 g, 2.269 mmol, 1.0 eq) at0° C. and the resulting reaction mixture was stirred at room temperaturefor 16 h as progress of the reaction was monitored by TLC (10% Methanolin DCM, Visualization: PMA). The reaction mixture was concentrated underreduced pressure to afford propyl 1,4-diazepane-1-carboxylatehydrochloride salt (0.41 g) as pale yellow gummy solid. MS (ESI): m/z187.12 [M+H]⁺.

Synthesis of propyl 4-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1,4-diazepane-1-carboxylate

To a stirred solution of 2-chloro-N-(2,6-dimethylphenyl) acetamide (0.4g, 2.023 mmol) in ACN (10 ml) was added and DIPEA (0.522 g, 4.046 mmol)and propyl 1,4-diazepane-1-carboxylate hydrochloride salt (0.376 g,2.023 mmol) at 0° C. The resulting reaction mixture was stirred at 90°C. for 16 h as progress of the reaction was monitored by TLC (50%EtOAc-Hexane, Visualization: UV). The reaction mixture was concentratedunder reduced pressure to afford residue which was diluted with EtOAc(100 ml) and washed with water (60 ml), dried over Na₂SO₄ andconcentrated under reduced pressure. The resulting crude product waspurified by normal phase flash chromatography (eluted with 10%-50% ofEtOAc in Pet ether) to afford propyl 4-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1,4-diazepane-1-carboxylate (0.25 g) as a whitesolid. MS (ESI): m/z 348.69 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.19(s, 1H), 7.00 (s, 3H), 3.96-3.91 (m, 2H), 3.50-3.43 (m, 4H), 3.25 (s,2H), 2.80-2.65 (m, 4H), 2.13 (s, 6H), 1.84-1.81 (m, 2H), 1.60-1.53 (m,2H), 0.90-0.85 (m, 3H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(propoxycarbonyl)-1,4-diazepan-1-ium bromide

To a stirred solution of propyl 4-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1,4-diazepane-1-carboxylate (0.25 g, 0.719 mmol) inACN (5 ml) was added benzyl bromide (0.419 g, 2.876 mmol) at roomtemperature and the resulting reaction mass was stirred at 90° C. for 16h as progress of the reaction was monitored by TLC (10% Methanol in DCM,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude product, which was triturated with Ethylacetate (40 ml) to afford 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(propoxycarbonyl)-1,4-diazepan-1-ium bromide (122.1mg) as a white solid. Mass (ESI): m/z 438.2 [M]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 9.99 (s, 1H), 7.61-7.53 (m, 5H), 7.18-7.11 (m, 3H), 5.00(s, 2H), 4.29-4.16 (m, 2H), 4.01-3.56 (m, 10H), 2.32-2.30 (m, 2H), 2.21(s, 6H), 1.62-1.57 (m, 2H), 0.92-0.88 (m, 3H).

Synthesis of1-benzyl-1-(2-((2-fluoro-6-methylphenyl)amino)-2-oxoethyl)azepan-1-iumbromide

Synthesis of 2-bromo-N-(2-fluoro-6-methylphenyl)acetamide

To a solution of 2-fluoro 6-methyl aniline (3.0 g, 23.980 mmol) in water(30.0 ml) was cooled to 0° C. and bromo acetyl bromide (29 g, 143.884mmol) was added. The resulting reaction mixture was stirred for 16 h atrt as progress of the reaction was monitored by TLC (50% EtOAc in petether, Visualization: UV). The reaction mixture was basified with NaCO₃,stirred for 20 min, filtered, washed with water (100 ml) followed by petether (50 mL) and dried to afford crude compound2-bromo-N-(2-fluoro-6-methylphenyl)acetamide (2.8 g) as an off whitesolid MS (ESI): m/z 246.08 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.95(s, 1H), 7.18 (d, 3H), 4.15 (s, 2H), 2.20 (s, 3H).

Synthesis of 2-(azepan-1-yl)-N-(2-fluoro-6-methylphenyl)acetamide

To a stirred solution of 2-bromo-N-(2-fluoro-6-methylphenyl) acetamide(4.0 g, 16.254 mmol) in ACN (40 ml) was added potassium carbonate (2.243g, 48.762 mmol) at room temperature and the mixture stirred for 10 min.Next, azepane (3.225 g, 32.509 mmol) was added and the reaction mixturewas stirred for 16 h at room temperature as progress of the reaction wasmonitored by TLC (50% EtOAc in pet ether, Visualization: UV). Thereaction mixture was diluted with ethyl acetate (100 ml), washed withwater (2×50 ml), dried over sodium sulphate, filtered and concentratedunder reduced pressure. Crude compound was triturated with n-pentane(3×30 ml) to afford 2-(azepan-1-yl)-N-(2-fluoro-6-methylphenyl)acetamide (2.2 g) as an off-white solid. MS (ESI): m/z 264.34 [M]⁺. ¹HNMR (400 MHz, CHLOROFORM-d) δ ppm 8.88 (s, 1H), 7.26 (s, 1H), 7.10-7.16(m, 1H), 6.93-7.03 (m, 1H), 3.31 (s, 2H), 2.82 (t, 4H), 2.27 (s, 3H),1.58-1.73 (m, 8H).

Synthesis of1-benzyl-1-(2-((2-fluoro-6-methylphenyl)amino)-2-oxoethyl)azepan-1-iumbromide

To a stirred solution of2-(azepan-1-yl)-N-(2-fluoro-6-methylphenyl)acetamide (0.50 g, 1.89 mmol)in ACN (5.0 mL) was added benzyl bromide (1.9411 g, 11.35 mmol) at roomtemperature and the resulting reaction mixture was heated to 80° C. for16 h as progress of the reaction was monitored by TLC (10% MeOH in DCM,Visualization: UV). The reaction mixture was cooled to room temperatureand concentrated under reduced pressure to afford crude compound whichwas triturated with a solution of 1:1 EtOAc/pet ether (3×10 mL) toafford product1-benzyl-1-(2-((2-fluoro-6-methylphenyl)amino)-2-oxoethyl)azepan-1-iumbromide (200 mg). MS (ESI): m/z 355.2 [M]⁺. ¹H NMR (400 MHz, DMSO-d6) δppm 10.19 (s, 1H), 7.64 (t, 2H), 7.51-7.57 (m, 3H), 7.27-7.32 (m, 1H),7.17 (t, 2H), 4.88 (s, 2H), 4.11 (s, 2H), 3.78 (q, 2H), 3.51-3.56 (m,2H), 2.27 (s, 3H), 1.96 (s, 4H), 1.66 (s, 4H).

The following examples were prepared from bromo acetyl bromide, benzylbromide, azepane and the appropriate aniline following proceduresdescribed for the synthesis of compound 69A.

MS Com- (ESI): pound # Structure m/z 70A

365.2 [M]⁺ 71A

367.3 [M]⁺ 72A

362.2 [M]⁺ 73A

405.2 [M]⁺ 74A

379.2 [M]⁺ 75A

379.2 [M]⁺ 76A

413.2 [M]⁺ 77A

381.3 [M]⁺ 78A

408.2 [M]⁺

Synthesis of 1-benzyl-1-(2-((2-hydroxy-6-methylphenyl)amino)-2-oxoethyl) azepan-1-ium formate

1-benzyl-1-(2-((2-hydroxy-6-methylphenyl) amino)-2-oxoethyl)azepan-1-ium

To a stirred solution of 1-benzyl-1-(2-((2-methoxy-6-methylphenyl)amino)-2-oxoethyl) azepan-1-ium bromide (1 g, 2.235 mmol) in DCM (25 ml)was added BBr₃ (2.239 g, 8.94 mmol) at 0° C. The resulting reactionmixture was stirred for 16 h at room temperature as progress of thereaction mixture was monitored by TLC (10% MeOH in DCM, Visualization:UV). The reaction mixture was concentrated under reduced pressure toafford crude product which was purified by normal phase flashchromatography (eluted with 6% MeOH in DCM). gradient. Pure fractionswere concentrated under reduced pressure to afford1-benzyl-1-(2-((2-hydroxy-6-methylphenyl) amino)-2-oxoethyl)azepan-1-ium bromide (0.9 g) as an off white solid. MS (ESI): m/z 353.21[M]⁺. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.82 (s, 1H), 9.59 (s, 1H),7.73-7.71 (m, 2H), 7.59-7.49 (m, 3H), 7.07-7.02 (t, 1H), 6.77-6.70 (m,2H), 4.88 (s, 2H), 4.06 (s, 2H), 3.77-3.72 (m, 2H), 3.58-3.53 (m, 2H),2.17 (s, 3H), 2.05-1.9 (m, 4H), 1.66-1.45 (m, 4H).

Synthesis of 1-benzyl-1-(2-((2-methyl-6-(propionyloxy) phenyl)amino)-2-oxoethyl) azepan-1-ium bromide

Synthesis of I-benzyl-1-(2-((2-methyl-6-(propionyloxy) phenyl)amino)-2-oxoethyl) azepan-1-ium bromide

Propionic anhydride (5 ml) was added to1-benzyl-1-(2-((2-hydroxy-6-methylphenyl) amino)-2-oxoethyl)azepan-1-ium bromide (0.4 g, 0.922 mmol) at room temperature and theresulting reaction mixture was heated to 80° C. for 16 h as progress ofthe reaction mixture was monitored by TLC (10% MeOH in DCM,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude product which was purified by normal phaseflash chromatography (eluted with 15% MeOH in DCM). Pure fractions wereconcentrated under reduced pressure to afford product which was againtriturated with diethyl ether (45 ml) to afford1-benzyl-1-(2-((2-methyl-6-(propionyloxy) phenyl) amino)-2-oxoethyl)azepan-1-ium bromide (95.6 mg) as an off white solid. MS (ESI): m/z409.2 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.16 (s, 1H), 7.66-7.64 (m,2H), 7.57-7.51 (m, 3H), 7.31-7.27 (t, 1H), 7.22-7.21 (d, 1H), 7.09-7.07(m, 1H), 4.89 (s, 2H), 4.08 (s, 2H), 3.75-3.71 (m, 2H), 3.51-3.47 (m,2H), 2.58 (q, 2H), 2.27 (s, 3H), 2.05-1.85 (m, 4H), 1.75-1.55 (m, 4H),1.12 (t, 3H).

Synthesis of 1-benzyl-1-(2-((2-carbamoyl-6-methylphenyl)amino)-2-oxoethyl) azepan-1-ium bromide

Synthesis of N, 3-trimethyl-2-nitrobenzamide

To a stirred solution of 3-methyl-2-nitrobenzoic acid (3.0 g, 16.56mmol) in DMF (30 ml) was added DIPEA (6.42 g, 49.68 mmol) and HATU (9.44g, 24.84 mmol) followed by dimethyl amine (2 M in THF) (16.56 ml, 33.12mmol). The resulting reaction mixture was stirred for 16 h at roomtemperature as progress of the reaction was monitored by TLC (30%EtOAc/pet ether, visualisation: UV). The reaction mixture was pouredinto ice water and extracted with EtOAc (2×50 ml). The combined extractswere was washed with brine (30 ml), dried over Na₂SO₄ and concentratedunder reduced pressure to afford the crude product which purified bynormal phase flash chromatography (eluted with 20%-80% of EtOAc in petether) to afford N, N, 3-trimethyl-2-nitrobenzamide (2.2 g) as blackliquid. Mass (ESI): m/z 209.09 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm7.47-7.43 (m, 1H), 7.36-7.33 (m, 1H), 7.22-7.19 (m, 1H), 3.09 (s, 3H),2.93 (s, 3H), 2.80 (s, 6H), 2.33 (s, 3H).

Synthesis of 2-amino-N, N, 3-trimethylbenzamide

To a stirred solution of N, N, 3-trimethyl-2-nitrobenzamide (2 g, 9.605mmol) in Ethanol (20 ml) and H₂O (20 ml) was added Fe (3.75 g, 67.235mmol) and NH₄Cl (3.59 g, 67.235 mmol) at room temperature. The resultingreaction mixture was stirred for 8 h at 80° C. as progress of thereaction was monitored by TLC (10% EtOAc in pet ether, visualization:UV). The reaction mixture was filtered through a celite pad and washedwith EtOH (2×50 ml) twice. Filtrate was concentrated under reducedpressure to afford residue which was diluted with water (50 ml) andextracted with EtOAc (2×50 ml). The combined organic extracts werewashed with brine (50 ml), dried over Na₂SO₄ and concentrated underreduced pressure to afford crude product which was purified by columnchromatography (eluted with 10%-40% of EtOAc in pet ether) to afford2-amino-N, N, 3-trimethylbenzamide (600 mg). LCMS purity: 98.02%, Mass(ESI): m/z 179.12 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.26 (s, 1H),7.06 (s, 1H), 6.97 (d, 1H), 6.66-6.63 (m, 1H), 4.33 (bs, 2H), 3.05 (s,6H), 2.17 (s, 3H).

Synthesis of 2-(2-bromoacetamido)-N, N, 3-trimethylbenzamide

To a stirred solution of isopropyl 2-amino-N, N, 3-trimethylbenzamide(0.6 g, 3.366 mmol) in H₂O (6 ml) was added 2-bromoacetyl bromide (5.34g, 26.928 mmol) at 0° C.

The resulting reaction mixture was stirred for 16 h at room temperatureas progress of the reaction was monitored by TLC (10% MeOH in DCM,Visualization: UV). The reaction mixture was basified with saturatedNa₂CO₃ at 0° C. to afford precipitated solid which was filtered, washedwith water and dried to afford (2-(2-bromoacetamido)-N, N,3-trimethylbenzamide (650 mg) as white solid. LCMS purity: 93.98%, Mass(ESI): m/z 301.12 [M+2]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.84 (s, 1H),7.26-7.07 (m, 3H), 3.93 (s, 2H) 3.09 (s, 3H), 2.93 (s, 3H), 2.21 (s,3H).

Synthesis of 2-(2-(azepan-1-yl) acetamide)-N, N, 3-trimethylbenzamide

To a stirred solution of 2-(2-bromoacetamido)-N, N, 3-trimethylbenzamide(0.6 g, 2.005 mmol) in Acetonitrile (6 ml) was added K₂CO₃ (0.692 g,5.012 mmol) and Azepane (0.397 g, 4.01 mmol) at room temperature. Theresulting reaction mixture was stirred for 16 h at 80° C. as progress ofthe reaction was monitored by TLC (10% MeOH in DCM, Visualization: UV).After completion of reaction on TLC, the reaction mixture wasconcentrated under reduced pressure to afford residue which was pouredinto ice water and extracted with EtOAc (2×25 ml). The combined organicextracts were washed with brine (50 ml), dried over Na₂SO₄ andconcentrated under reduced pressure to afford (2-(2-(azepan-1-yl)acetamido)-N, N,3-trimethylbenzamide (600 mg). MS (ESI): m/z 318.27[M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 9.19 (s, 1H) 7.27-7.25 (m, 1H),7.20-7.17 (m, 1H), 7.09-7.07 (m, 1H), 3.24 (s, 2H) 3.04 (s, 3H), 2.91(s, 3H), 2.79-2.76 (t, 4H), 2.26 (s, 3H) 1.71-1.62 (m, 8H).

Synthesis of 1-benzyl-1-(2-((2-(dimethylcarbamoyl)-6-methylphenyl)amino)-2-oxoethyl) azepan-1-ium bromide

To a stirred solution of2-(2-(azepan-1-yl)acetamido)-N,N,3-trimethylbenzamide (0.5 g, 1.575mmol) in acetonitrile (5 ml) was added benzyl bromide (0.538 g, 3.15mmol) at room temperature. The resulting reaction mixture was stirredfor 48 h at 80° C. as progress of the reaction was monitored by TLC(Mobile phase: −10% MeOH in DCM, Rf: 0.47, Visualization: UV). Afterconsumption of starting material on TLC, the reaction mixture wasconcentrated under reduced pressure to afford crude product which waspurified by column chromatography (eluted with 4%-8% of MeOH in DCM) toafford the (1-benzyl-1-(2-((2-(dimethylcarbamoyl)-6-methylphenyl)amino)-2-oxoethyl) azepan-1-ium bromide (100 mg) as a brown solid. Mass(ESI): m/z 408.2 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.31 (s, 1H),7.70-7.68 (m, 2H), 7.56-7.51 (m, 3H), 7.38-7.36 (m, 1H), 7.32-7.28 (m,1H), 7.19-7.16 (m, 1H), 4.85 (s, 2H), 4.03 (s, 2H), 3.70-3.66 (m, 2H),3.47-3.43 (m, 2H), 2.95 (s, 3H), 2.87 (s, 3H), 2.27 (s, 3H), 1.94-1.90(s, 4H), 1.70-1.65 (s, 4H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylbenzoyl) oxy) ethyl)azepan-1-ium bromide

Synthesis of Intermediate 2-bromoethyl 2,6-dimethylbenzoate

To a stirred solution of 2,6-dimethylbenzoic acid (5 g, 33.29 mmol) intoluene (40 mL) was added 2-bromoethan-1-ol (4.16 g, 33.29 mmol) and acatalytic amount of concentrated H₂SO₄ (0.2 ml). The resulting reactionmixture was refluxed for 16 h using a Dean-Stark condenser as progressof the reaction was monitored by TLC (30% EtOAc in Pet Ether,visualization: UV). After completion reaction, the mixture was cooled toroom temperature and concentrated under reduced pressure to affordresidue which was diluted with ice water (250 ml) and extracted withEtOAc (3×150 ml). The combined organic extracts were washed withsaturated sodium bicarbonate solution (50 ml) and brine (100 ml), driedover anhydrous sodium sulphate and concentrated under reduced pressureto afford 2-bromoethyl 2,6-dimethylbenzoate (5.52 g). MS (ESI): m/z258.82 [M+H+2]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.22-7.18 (m, 1H),7.0-7.02 (m, 2H), 4.64 (t, 2H), 3.63 (t, 2H), 2.33 (s, 6H).

Synthesis of Intermediate 2-(azepan-1-yl) ethyl 2,6-dimethylbenzoate

To a stirred solution of 2-bromoethyl 2,6-dimethylbenzoate (2.0 g, 7.778mmol) in ACN (30 ml) was added K₂CO₃ (3.224 g, 23.334 mmol) and azepane(1.157 g, 11.667 mmol).

The resulting mixture was stirred at 90° C. for 16 h as progress of thereaction was monitored by TLC (30% EtOAc in pet ether, Visualization:UV.) The reaction mixture was allowed to cool to room temperature andconcentrated under reduced pressure to afford residue which was dilutedwith cold water (100 ml) and extracted with EtOAc (3×50 ml). Thecombined organic extracts were was washed with brine (50 ml), dried overanhydrous sodium sulphate and concentrated under reduced pressure toafford 2-(azepan-1-yl)ethyl 2,6-dimethylbenzoate (1.80 g) as a brownliquid. MS (ESI): m/z 276.35 [M+H]+¹H NMR (400 MHz, CDCl₃) δ ppm7.19-7.15 (m, 1H), 7.03-7.01 (m, 2H), 4.41 (t, 2H), 2.86 (t, 2H),2.72-2.69 (mt, 4H), 2.33 (s, 6H), 1.65-1.56 (m, 8H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylbenzoyl) oxy) ethyl)azepan-1-ium bromide

To a stirred solution of 2-(azepan-1-yl) ethyl 2,6-dimethylbenzoate (300mg, 1.089 mmol) in ACN (5 ml) was added benzyl bromide (279.3 mg, 1.633mmol) and the resulting mixture was stirred at 90° C. for 16 h asprogress of the reaction was monitored by TLC (MeOH in DCM,visualization: UV). After consumption of starting material, the reactionmixture was allowed to cool to room temperature and then concentratedunder reduced pressure to afford crude product, which was trituratedwith a 1:2 mixture of Et₂₀ & EtOAc (60 ml) to afford1-benzyl-1-(2-((2,6-dimethylbenzoyl) oxy) ethyl) azepan-1-ium bromide(117.4 mg,) as an off white solid. Mass (ESI): m/z 366.2 [M]⁺. ¹H NMR(400 MHz, DMSO-d₆) δ ppm 7.61-7.59 (m, 2H), 7.54-7.48 (m, 3H), 7.29-7.26(m, 1H), 7.13-7.11 (d, 2H), 4.89 (t, 2H), 4.67 (s, 2H), 3.65-3.45 (m,6H), 2.28 (s, 6H), 1.83-.1.77 (d, 4H), 1.6-1.5 (m, 4H).

Synthesis of 1-benzyl-1-(2-(2,6-dimethylphenoxy)-2-oxoethyl)azepan-1-ium 2,2,2-trifluoroacetate

Synthesis of Intermediate 2,6-dimethylphenyl 2-bromoacetate

To a stirred solution of 2,6-dimethyl phenol (0.5 g, 4.092 mmol) in ACN(5 ml) was added pyridine (0.647 g, 8.184 mmol) and 2-bromoacetylbromide (1.23 g, 6.138 mmol) at 0° C. and the resulting reaction mixturewas stirred at 0° C. for 15 min as progress of the reaction wasmonitored by TLC (10% EtOAc in Pet ether, Visualization: UV). Afterconsumption of starting, the reaction mixture was diluted with water (20ml) and extracted with EtOAc (2×25 ml). The combined organic extractswere washed with brine (30 ml), dried over Na₂SO₄ and concentrated underreduced pressure to afford product 2,6-dimethylphenyl 2-bromoacetate(500 mg) as a pale yellow liquid. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.07 (s,3H), 4.07 (s, 2H), 2.18 (s, 6H).

Synthesis of Intermediate 2,6-dimethylphenyl 2-(azepan-1-yl) acetate

To a stirred solution of 2,6-dimethylphenyl 2-bromoacetate (0.5 g, 2.056mmol) in ACN (5 ml) was added K₂CO₃ (0.71 g, 5.14 mmol) and azepane(0.407 g, 4.112 mmol) at room temperature. The resulting reactionmixture was stirred at 90° C. for 16 h as progress of the reaction wasmonitored by TLC (Mobile phase: Ethyl acetate, Visualization: UV). Aftercompletion of reaction, the mixture was concentrated under reducedpressure to afford residue which was diluted with ice water (25 ml) andextracted with ethyl acetate (2×100 ml). The combined organic extractswere washed with brine (50 ml), dried over Na₂SO₄ and concentrated underreduced pressure to afford 2,6-dimethylphenyl 2-(azepan-1-yl) acetate(500 mg) as red liquid. Mass (ESI): m/z 262.09 [M+H]⁺.

Synthesis of 1-benzyl-1-(2-(2,6-dimethylphenoxy)-2-oxoethyl)azepan-1-ium 2,2,2-trifluoroacetate

To a stirred solution of 2,6-dimethylphenyl 2-(azepan-1-yl) acetate(0.45 g, 1.721 mmol) in ACN (4.5 ml) was added benzyl bromide (0.588 g,3.442 mmol) and the resulting reaction mixture was stirred at 90° C. for16 h as progress of the reaction was monitored by TLC (Mobile phase: 10%MeOH in DCM, Visualization: UV). After completion of reaction, thereaction mixture was concentrated under reduced pressure to afford thecrude product which was purified by the reverse phase Prep HPLC (Column:X select phenyl hexyl C18 (19*250), Sum; Mobile phase A: 0.1% TFA (Aq);Mobile phase B-Acetonitrile; Flow: 15 ml/min; Method: 0/20, 2/20, 10/50,15/75, 15.2/98, 19/98, 19.2/20, 23/20; Solubility: ACN+Water+THF;Temperature: ambient).

The collected pure fractions were lyophilized to afford1-benzyl-1-(2-(2,6-dimethylphenoxy)-2-oxoethyl) azepan-1-ium2,2,2-trifluoroacetate (56 mg) as pale yellow solid. MS (ESI): m/z 352.2[M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.60-7.53 (m, 5H), 7.21-7.16 (m,3H), 4.87 (s, 2H), 4.71 (s, 2H), 3.90-3.84 (m, 2H), 3.66-3.60 (m, 3H),2.19 (s, 6H), 2.07-1.91 (m, 4H), 1.40-1.20 (s, 4H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) amino) ethyl)azepan-1-ium trifluoroacetate

Synthesis of Intermediate 1-(azepan-1-yl)-2-chloroethan-1-one

To a cooled solution of azepane (10 g, 100.826 mmol) (0° C.) in ACN (100mL) was added TEA (30.6 g, 302.478 mmol) followed by 2-chloroacetylchloride (13.6 g, 120.98 mmol). The reaction mixture was stirred at roomtemperature for 3 h as progress of the reaction was monitored by TLC(Mobile phase: EtOAc, visualization by UV and ninhydrin). The reactionwas quenched with saturated aq. NaHCO₃ and extracted with EtOAc (2×250mL).

The combined organic extracts were washed with brine (200 mL), driedover anhydrous Na₂SO₄, filtered, and concentrated under reduced pressureto afford crude product which was purified by column chromatography(eluted with 30% EtOAc in pet ether) to afford1-(azepan-1-yl)-2-chloroethan-1-one (8.5 g) as a brown liquid. Mass(ESI): m/z 175.89 [M+1]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 4.08 (s, 2H),3.49-3.55 (m, 4H), 1.72-1.79 (m, 4H), 1.58-1.63 (d, 4H).

Synthesis of Intermediate 1-(azepan-1-yl)-2-((2,6-dimethylphenyl) amino)ethan-1-one

To a stirred solution of 1-(azepan-1-yl)-2-chloroethan-1-one (5 g,28.464 mmol) in ACN (40 mL) was added DIPEA (11.03 g, 85.392 mmol)followed by 2,6-dimethylaniline (5.17 g, 42.696 mmol) and the resultingreaction mixture was stirred at 90° C. for 3 days as progress of thereaction was monitored by TLC (Mobile phase: 30% EtOAc in pet-ether,Visualization: UV). The reaction mixture was quenched with saturated aq.NaHCO₃ (20 ml) and extracted with EtOAc (2×250 ml). The combined organicextracts were washed with brine (200 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford crude product(8.5 g) which was purified by column chromatography (eluted with 30% ofEtOAc in pet-ether) to afford 1-(azepan-1-yl)-2-((2,6-dimethylphenyl)amino) ethan-1-one (2.7 g, 36%) as a brown solid. Mass (ESI): m/z 261.18[M+1]⁺. ¹H NMR (400 MHz, cdcl3) δ ppm 6.97-7.99 (m, 2H), 6.76-6.80 (m,1H), 4.78 (s, 1H), 3.81 (s, 2H), 3.33-3.58 (m, 2H), 3.30-3.32 (m, 2H),2.34 (s, 6H), 1.72-1.73 (m, 4H), 1.55-1.59 (m, 5H).

Synthesis of Intermediate N-(2-(azepan-1-yl) ethyl)-2,6-dimethylaniline

A solution of 1-(azepan-1-yl)-2-((2,6-dimethylphenyl) amino) ethan-1-one(1.5 g, 5.760 mmol) in THE (15 mL) was cooled to 0° C. and LAH (2 M inTHF, 5.76 mL, 11.521 mmol) was added drop-wise with stirring. Theresulting mixture was then stirred at room temperature for 15 h asprogress of the reaction was monitored by TLC (Mobile phase: 30% EtOAcin pet-ether, Visualization: UV). The reaction mixture was quenched withsaturated aq. NH₄Cl solution at 0° C., and then allowed to stir at RTfor 1 h. The precipitated solid was removed by filtration and thefiltrate was concentrated under reduced pressure to affordN-(2-(azepan-1-yl) ethyl)-2,6-dimethylaniline (1.4 g) as a pale yellowliquid. Mass (ESI): m/z 247.23 [M+1].

Synthesis of Intermediate t-butyl (2-(azepan-1-yl) ethyl)(2,6-dimethylphenyl) carbamate

To a stirred solution of N-(2-(azepan-1-yl) ethyl)-2,6-dimethylaniline(800 mg, 3.247 mmol) in a 1:1 mixture of 1,4-dioxane and H₂O (10 ml) wasadded NaOH (260 mg, 6.494 mmol) followed by (Boc)₂O (2.12 g, 9.741 mmol)and the resulting mixture was stirred at rt for 24 h as progress of thereaction was monitored by TLC (Mobile phase: 30% EtOAc in pet-ether,detection: ninhydrin). The reaction mixture was diluted with water (75mL), extracted with EtOAc (2×75 mL) and the combined organic extractswere washed with brine (70 mL), dried over anhydrous Na₂SO₄, filtered,and concentrated under reduced pressure. The crude product was purifiedby column chromatography (eluted with 15% of EtOAc in pet-ether) toafford tert-butyl (2-(azepan-1-yl) ethyl) (2,6-dimethylphenyl) carbamate(600 mg) as a colour less liquid. Mass (ESI): m/z 347.24 [M+1]⁺. ¹H NMR(400 MHz, DMSO-d₆) δ ppm 7.00-7.26 (m, 3H), 3.47-3.57 (m, 2H), 2.60-2.73(m, 6H), 2.30 (s, 6H), 2.22 (t, 11H), 1.57 (s, 6H).

Synthesis of Intermediate 1-benzyl-1-(2-((tert-butoxycarbonyl)(2,6-dimethylphenyl) amino) ethyl) azepan-1-ium chloride

To a stirred solution of tert-butyl (2-(azepan-1-yl)ethyl)(2,6-dimethylphenyl) carbamate (300 mg, 0.865 mmol) in ACN (4 mL)was added benzyl chloride (328 mg, 2.595 mmol) and the resultingreaction mixture was stirred at 90° C. for 15 h as progress of thereaction was monitored by TLC (Mobile phase: 10% MeOH in DCM,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude 1-benzyl-1-(2-((tert-butoxycarbonyl)(2,6-dimethylphenyl) amino) ethyl) azepan-1-ium chloride salt (400 mg)as a colour less gum. Mass (ESI): m/z 437.30 [M+1]⁺.

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) amino) ethyl)azepan-1-ium trifluoro acetate

To a stirred solution of 1-benzyl-1-(2-((tert-butoxycarbonyl)(2,6-dimethylphenyl) amino) ethyl) azepan-1-ium chloride (360 mg, 0.634mmol) was added 4 M HCl in 1,4-dioxane (3.17 mL, 12.680) at 0° C. Theresulting reaction mixture was stirred at rt for 15 h as progress of thereaction was monitored by TLC (Mobile phase: 10% MeOH in DCM,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude product which was purified by reverse phasePrep.HPLC (Column: X-Bridge C18 (150*25) mm, 10u; Mobile phase A: 0.1%TFA in Water (Aq); Mobile phase B: Acetonitrile; Flow: 14 ml/min; Method(T/% of B): 0/20, 2/30, 10/40, 19/40, 19.2/98, 22/98, 22.2/20, 26/20;Solubility: ACN+WATER+THF; Temperature: Ambient). Pure fractionscollected and lyophilized to afford 1-benzyl-1-(2-((2,6-dimethylphenyl)amino) ethyl) azepan-1-ium trifluoro acetate (165 mg) as a pale brownsolid. Mass (ESI): m/z 337.30 [M]⁺. LCMS: 99.72%. ¹H NMR (400 MHz,DMSO-d₆) δ ppm 7.41-7.53 (m, 5H), 6.96-7.21 (m, 2H), 6.83 (t, 1H), 4.56(s, 2H), 3.31-3.53 (m, 8H), 2.27 (s, 6H), 1.82-1.83 (m, 4H), 1.58-1.59(m, 4H).

Synthesis of 1-benzyl-1-(2-(2,6-dimethylphenoxy) ethyl) azepan-1-iumbromide

Synthesis of 2-(2-bromoethoxy)-1,3-dimethylbenzene

To a stirred solution of 2,6-dimethylphenol (0.500 g, 4.096 mmol) inacetonitrile (8.0 mL) was added potassium carbonate (1.698 g, 12.288mmol) and 1,2-dibromoethane (3.847 g, 20.480 mmol). The resultingreaction mixture was stirred at 90° C. for 16 h as progress of thereaction was monitored by TLC (Mobile phase: 5% EtOAc in pet ether,Visualization: UV). The reaction mixture was cooled to RT andconcentrated under reduced pressure, diluted with water (100 mL) andextracted with dichloromethane (3×50 mL). The combined organic extractswere washed with brine solution (1×50 ml), dried over anhydrous sodiumsulphate and concentrated under reduced pressure to afford crude whichwas purified by silica gel chromatography (eluted with pet ether) toafford pure 2-(2-bromoethoxy)-1,3-dimethylbenzene (0.20 g) as acolourless liquid. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.00-7.25 (m, 2H),6.91-6.95 (m, 1H), 4.01-4.10 (m, 2H), 3.65-3.68 (m, 2H), 2.30 (s, 6H).

Synthesis of Intermediate 1-(2-(2,6-dimethylphenoxy) ethyl) azepane

To a solution of 2-(2-bromoethoxy)-1,3-dimethylbenzene (200 mg, 0.87mmol) in acetonitrile (3.0 mL) was added DIPEA (451.2 mg, 3.49 mmol) andazepane (120.2 mg, 1.22 mmol). The resulting reaction mixture wasstirred at 90° C. for 16 h as progress of the reaction was monitored byTLC. (Mobile phase: 10% EtOAc in pet ether, Visualization: UV). Thereaction mixture was cooled to RT and concentrated under reducedpressure, diluted with water (20 mL) and extracted with dichloromethane(3×20 mL). The combined organic extracts were washed with brine solution(1×20 ml), dried over anhydrous sodium sulphate and concentrated underreduced pressure to afford 1-(2-(2,6-dimethylphenoxy) ethyl) azepane(210 mg). ¹H NMR (400 MHz, CDCl₃) δ ppm 6.98-7.00 (m, 2H), 6.88-6.92 (m,1H), 3.85-3.88 (m, 2H), 2.93-2.96 (m, 2H), 2.75-2.77 (m, 4H), 2.28 (s,6H), 1.59-1.68 (m, 8H).

Synthesis of 1-benzyl-1-(2-(2,6-dimethylphenoxy) ethyl) azepan-1-iumbromide

To a solution of 1-(2-(2,6-dimethylphenoxy) ethyl) azepane (100 mg,0.404 mmol) in acetonitrile (1.5 mL) was added benzyl bromide (0.072 ml,0.606 mmol) and the resulting reaction mixture was stirred at 90° C. for16 h in sealed tube as progress of the reaction was monitored by TLC(Mobile phase: 50% EtOAc in pet ether, Visualization: UV). The reactionmixture was cooled to room temperature and concentrated under reducedpressure to afford crude gummy which was triturated with ethyl acetate(3×5 ml) to afford 1-benzyl-1-(2-(2,6-dimethylphenoxy) ethyl)azepan-1-ium bromide (80.5 mg) as an off white solid. MS (ESI): m/z338.41 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.63-7.65 (m, 2H),7.49-7.57 (m, 3H), 7.04-7.08 (m, 2H), 6.96-6.98 (m, 1H), 4.72 (s, 2H),4.29 (t, 2H), 3.52-3.69 (m, 6H), 2.29 (s, 6H), 1.88-1.92 (m, 4H),1.59-1.62 (m, 4H).

Synthesis of N-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)-N,N-diethyl-3-phenylpropan-1-aminium bromide

Synthesis of Intermediate N, N-diethyl-3-phenylpropan-1-amine

To a stirred solution of (3-bromopropyl) benzene (2 g, 10.04 mmol) inacetonitrile (5 ml) was added diethylamine (1.46 g, 19.96 mmol) at roomtemperature. The resulting reaction mixture stirred for 16 h at 75° C.as progress of the reaction was monitored by TLC (Mobile phase: 50%EtOAc in pet ether, Visualization by UV). The reaction mixture wasallowed to cool to room temperature, quenched with water and extractedwith ethyl acetate (2×15 ml). The combined organic extracts were driedover anhydrous sodium sulphate and concentrated under reduced pressureto afford N, N-diethyl-3-phenylpropan-1-amine (1.3 g). MS (ESI): m/z192.25 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.3-7.25 (m, 2H), 7.22-7.15(m, 3H), 2.6-2.25 (m, 2H), 2.55-2.42 (m, 6H), 1.81-1.75 (m, 2H), 1.0 (t,6H).

Synthesis of N-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)-N,N-diethyl-3-phenylpropan-1-aminium bromide

To a solution of N, N-diethyl-3-phenylpropan-1-amine (0.2 g, 1.045 mmol)in toluene (5 ml) was added 2-bromo-N-(2,6-dimethylphenyl) acetamide(0.328 g, 1.354 mmol) and the resulting mixture was heated to reflux for16 h as progress of the reaction was monitored by TLC (Mobile phase: 10%MeOH in DCM, visualization: UV). The reaction mixture was cooled to roomtemperature and concentrated under reduced pressure to afford crudeproduct which was triturated with EtOAc (20 ml) to affordN-(2-((2,6-dimethylphenyl) amino)-2-oxoethyl)-N,N-diethyl-3-phenylpropan-1-aminium bromide (95 mg) as an off whitesolid. MS (ESI): m/z 353.45 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.9(s, 1H), 7.45-7.3 (m, 5H), 7.15-7.09 (m, 3H), 4.28 (s, 2H), 3.58 (q,4H), 3.51-3.45 (m, 2H), 2.68-2.51 (m, 2H), 2.15 (s, 6H), 2.08-2.0 (m,2H), 1.28 (t, 6H).

Synthesis of N-benzyl-2-((2,6-dimethylphenyl) sulfonyl)-N,N-diethylethan-1-aminium bromide

Synthesis of Intermediate (2-chloroethyl) (2,6-dimethylphenyl) sulfane

To a stirred solution of 2,6-dimethylbenzenethiol (2 g, 14.468 mmol, 1eq) in ethanol (30 ml) was added NaOH (1.736 g, 43.405 mmol) and1-bromo-2-chloroethane (4.149 g, 28.937 mmol) at 0° C. The resultingreaction mixture was stirred at RT for 16 h as progress of the reactionmixture was monitored by TLC (Mobile phase: 100% Pet ether,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude residue which was diluted with ethyl acetate(80 ml), washed with water (50 ml×2) and brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to afford crud product.The crude product was purified by flash chromatography (eluted with Petether). The collected pure fractions were concentrated under reducedpressure to afford (2-chloroethyl) (2,6-dimethylphenyl) sulfane (0.500g) as a colourless liquid. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.085-7.163 (m,3H), 3.502-3.542 (m, 2H), 2.963-3.003 (m, 2H), 2.544 (s, 6H).

Synthesis of Intermediate 2-((2-chloroethyl)sulfonyl)-1,3-dimethylbenzene

To a stirred solution of (2-chloroethyl) (2,6-dimethylphenyl) sulfane(0.500 g, 2.491 mmol) in acetic acid (10 ml) was added hydrogen peroxide(33% in H₂O) (0.847 g, 24.910 mmol) at RT, and the reaction mixture washeated to 110° C. for 16 h as progress of the reaction was monitored byTLC (Mobile phase: 20% Ethyl acetate in pet ether, Visualization: UV).The reaction mixture was diluted with water (60 ml) and extracted withethyl acetate (70 ml×3). The combined organic extracts were washed withaq. sodium thiosulfate (50 ml×2) and brine solution (50 ml), and thenconcentrated under reduced pressure to afford crude product which waspurified by flash chromatography (eluted with 10% Ethyl acetate in Pet)The collected pure fractions were concentrated under reduced pressure toafford 2-((2-chloroethyl) sulfonyl)-1,3-dimethylbenzene (0.350 g) as anoff white solid. MS (ESI): m/z 232 [M]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm7.345-7.383 (m, 1H), 7.176-7.195 (m, 2H), 3.815-3.854 (m, 2H),3.531-3.569 (m, 2H), 2.714 (s, 6H).

Synthesis of Intermediate 2-((2,6-dimethylphenyl) sulfonyl)-N,N-diethylethan-1-amine

To a stirred solution of 2-((2-chloroethyl)sulfonyl)-1,3-dimethylbenzene (0.300 g, 1.289 mmol) in acetonitrile (10ml) was added potassium iodide (0.428 g, 2.578 mmol) and diethylamine(0.283 g, 3.867 mmol) at RT. The resulting reaction mixture was heatedto 90° C. for 16 h in a sealed tube as progress of the reaction mixturewas monitored by TLC (Mobile phase: 50% Ethyl acetate in pet ether,visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude product which was diluted with ethyl acetate(60 ml), washed with water (30 ml×3) and brine (30 ml), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford crudeproduct which was purified by flash chromatography (eluted with 40%ethyl acetate in Pet ether). The collected pure fractions wereconcentrated under reduced pressure to afford the desired product2-((2,6-dimethylphenyl) sulfonyl)-N, N-diethylethan-1-amine (0.200 g) asan off white solid. MS (ESI): m/z 270.16 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃)δ ppm 7.308-7.346 (m, 1H), 7.153-7.172 (m, 2H), 3.244-3.283 (m, 2H),2.947-2.985 (m, 2H), 2.716 (s, 6H), 2.437-2.490 (m, 4H), 0.937-0.973 (m,6H).

Synthesis of N-benzyl-2-((2,6-dimethylphenyl) sulfonyl)-N,N-diethylethan-1-aminium bromide

To a stirred solution of 2-((2,6-dimethylphenyl) sulfonyl)-N,N-diethylethan-1-amine (0.100 g, 0.371 mmol) in acetonitrile (5 ml) wasadded benzyl bromide (0.127 g, 0.742 mmol) and the resulting reactionmixture was heated to 90° C. for 16 h as progress of the reactionmixture was monitored by TLC (Mobile phase: 10% MeOH in DCM,Visualization: UV). The reaction mixture was concentrated under reducedpressure to afford crude which was triturated with EtOAc (20 ml) andn-pentane (10 ml) to afford N-benzyl-2-((2,6-dimethylphenyl)sulfonyl)-N, N-diethylethan-1-aminium bromide (0.060 mg) as an off whitesolid. MS (ESI): m/z 360.0 [M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm7.475-7.552 (m, 6H), 7.347-7.366 (d, 2H), 4.604 (s, 2H), 3.925-3.965 (m,2H), 3.520-3.560 (m, 2H), 3.243-3.288 (m, 4H), 2.676 (s, 6H),1.235-1.312 (m, 6H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) sulfonyl) ethyl)azepan-1-ium bromide

Synthesis of Intermediate (2-((2,6-dimethylphenyl) sulfonyl) ethyl)

To a stirred solution of 2-((2-chloroethyl)sulfonyl)-1,3-dimethylbenzene(1.0 g, 4.2970 mmol) in ACN (10.0 ml) was added azepane (0.639 g, 6.4455mmol) and potassium iodide (1.426 g, 8.5940 mmol). The resultingreaction mixture was heated to reflux for 16 h as progress of thereaction was monitored by TLC (Mobile phase: 30% EtOAc in pet ether,visualization: UV). The reaction mixture was diluted with ethyl acetate(50 mL) and washed with water (2×50 ml) and brine (1×50 mL). The organicphase was then, dried over anhydrous sodium sulphate, filtered andconcentrated under reduced pressure to afford crude compound which wastriturated with 2×10 ml of n-pentane to afford (2-((2,6-dimethylphenyl)sulfonyl) ethyl) azepane (180 mg) as an off white solid. MS (ESI): m/z295.44 [M+H]. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.34 (t, 1H), 7.17 (d, 2H),3.1-3.8 (m. 6H), 2.71 (d, 6H), 1.57-1.82 (m, 10H), 2.71 (d, 6H).

Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl) sulfonyl) ethyl)azepan-1-ium bromide

To a stirred solution of 1-(2-((2,6-dimethylphenyl) sulfonyl) ethyl)azepane (0.550 g, 1.864 mmol) in ACN (6.0 ml) was added benzyl bromide(0.637 g, 3.728 mmol) and the resulting reaction mixture was heated toreflux for 48 h as progress of the reaction was monitored by TLC (Mobilephase: 10% Methanol in DCM, Visualization: UV). The reaction mixture wascooled to room temperature and concentrated under reduced pressure toafford crude compound which was triturated with 3×10 ml of ethyl acetateto afford 1-benzyl-1-(2-((2,6-dimethylphenyl) sulfonyl) ethyl)azepan-1-ium bromide (80 mg) as an off white solid. MS (ESI): m/z 386.40[M]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.5-7.63 (m, 6H), 7.44 (d, 2H),4.59-4.65 (d, 2H), 4.06-4.15 (m, 2H), 3.49-3.55 (m, 6H), 2.696 (s, 6H),1.817-1.95 (m, 4H), 1.569-1.65 (m, 4H).

Synthesis of1-benzyl-1-(1-(2,6-dimethylphenyl)-2-oxopyrrolidin-3-yl)piperidin-1-iumbromide

Synthesis of 3-bromo-1-(2,6-dimethylphenyl) pyrrolidin-2-one

A stirred solution of 2,6-dimethylaniline (1.0 g, 8.252 mmol) in ACN (20ml) was treated with K₃PO₄ (1.751 g, 8.252 mmol) and 2,4-dibromobutanoylchloride (2.181 g, 8.252 mmol) at 0° C., and the reaction mixture wasstirred at RT for 1 h. After 1 h, the resulting reaction mixture wastreated with 50% NaOH aq. solution (1.650 g in 3.32 mL water) and thereaction mixture was stirred at RT for another 1 h as progress of thereaction mixture was monitored by TLC (Mobile phase: 50% Ethyl acetatein pet ether, Visualization: UV). After completion, the reaction mixturewas filtered to remove inorganic salts and the filtrate was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford crudeproduct as a colourless gummy. The crude product was triturated withn-pentane (30 ml) to afford 3-bromo-1-(2,6-dimethylphenyl)pyrrolidin-2-one (1.5 g). MS (ESI): m/z 268.03 [M+H]⁺. ¹H NMR (400 MHz,CDCl₃) δ ppm 7.18-7.14 (m, 1H), 7.10-7.08 (m, 2H), 4.56-4.54 (m, 1H),3.92-3.86 (m, 1H), 3.51-3.46 (m, 1H), 2.84-2.78 (m, 1H), 2.52-2.47 (m,1H), 2.29 (s, 3H), 2.19 (s, 3H).

Synthesis of 1-(2,6-dimethylphenyl)-3-(piperidin-1-yl) pyrrolidin-2-one

To a stirred solution of 3-bromo-1-(2,6-dimethylphenyl) pyrrolidin-2-one(0.300 g, 1.118 mmol) in ACN (10 ml) was added piperidine (0.114 g,1.342 mmol) and DIPEA (0.578 g, 4.475 mmol) at RT and the resultingmixture was heated to 90° C. for 16 h in a sealed tube as progress ofthe reaction mixture was monitored by TLC (Mobile phase: 10% Methanol inDCM, Visualization: UV). The reaction mixture was concentrated underreduced pressure to afford crude product which was diluted with DCM (70ml) and washed with water (40 ml×3) and brine (40 ml). The organic layerwas dried over anhydrous Na₂SO₄ and concentrated under reduced pressureto afford 1-(2,6-dimethylphenyl)-3-(piperidin-1-yl) pyrrolidin-2-one(0.220 g). MS (ESI): m/z 273.35 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ ppm7.14-7.06 (m, 3H), 3.70-3.48 (m, 3H), 2.80-2.89 (m, 2H), 2.60-2.65 (m,2H), 2.35-2.28 (m, 2H), 2.21 (s, 3H), 2.15 (s, 3H), 1.67-1.60 (m, 4H),1.50-1.46 (m, 2H).

Synthesis of 1-benzyl-1-(1-(2,6-dimethylphenyl)-2-oxopyrrolidin-3-yl)piperidin-1-ium bromide

To a stirred solution of 1-(2,6-dimethylphenyl)-3-(piperidin-1-yl)pyrrolidin-2-one (0.220 g, 0.807 mmol) in ACN (5 ml) was added benzylbromide (0.276 g, 1.615 mmol) and the resulting mixture was heated at90° C. for 16 h in a sealed tube as progress of the reaction mixture wasmonitored by TLC (Mobile phase: 10% Methanol in DCM, Visualization: UV).The reaction mixture was cooled to RT, diluted with ethyl acetate (20ml) and stirred for 2 h to afford solid which was filtered and washedwith ethyl acetate (40 ml) followed by n-pentane (20 ml) and dried underhigh vacuum to afford1-benzyl-1-(1-(2,6-dimethylphenyl)-2-oxopyrrolidin-3-yl) piperidin-1-iumbromide (0.090 g) as an off white solid. MS (ESI): m/z 363.1 [M]⁺. ¹HNMR (400 MHz, DMSO-d6) δ ppm 7.60-7.52 (m, 5H), 7.25-7.16 (m, 3H),5.58-4.02 (m, 4H), 3.65-3.57 (m, 2H), 3.13-2.92 (m, 3H), 2.67-2.66 (m,1H), 2.21-1.94 (m, 10H), 1.61-1.23 (m, 3H).

The following example was prepared from 3-bromo-1-(2,6-dimethylphenyl)pyrrolidin-2-one, azepane and benzyl bromide following proceduresdescribed for the synthesis of compound 89A.

Compound # Structure MS (ESI): m/z 90A

391.44 [M]⁺

Synthesis of 1-benzyl-1-(1-(2,6-dimethylphenyl)-2-oxopiperidin-3-yl)pyrrolidin-1-ium bromide

Synthesis of 2,5-dibromopentanoyl chloride

To a stirred suspension of 5-bromopentanoyl chloride (5 g, 25.066 mmol)was treated with bromine (2.56 ml, 50.132 mmol) at room temperature andthe resulting reaction mixture was then stirred at 100° C. for 2 hours.After 2 h, the reaction was cooled to room temperature and concentratedunder reduced pressure to afford crude 2,5-dibromopentanoyl chloride(7.2 g) which was carry forwarded to next step without furtherpurification.

Synthesis of 3-bromo-1-(2,6-dimethylphenyl) piperidin-2-one

A stirred solution of 2,6-dimethylaniline (2.0 g, 16.504 mmol) andpotassium phosphate (3.503 g, 16.504 mmol) in acetonitrile (50 ml) wascooled to 0° C. and 2,5-dibromopentanoyl chloride (4.594 g, 16.504 mmol)was added dropwise. The resulting reaction mixture was stirred at roomtemperature for 1 h. After 1 h, 50% NaOH aq. solution (3.3 g, 6.6 mlwater) was added to the reaction mixture and the resulting reactionmixture was stirred at room temperature for 1 h as progress of thereaction was monitored by TLC (Mobile phase: 50% EtOAc:Pet ether,Visualization: UV). The reaction mixture was filtered to removeinorganic salts and the filtrate was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford crude product which waspurified by column chromatography (eluted with 25%-30% of EtOAc in Petether) to afford 3-bromo-1-(2,6-dimethylphenyl) piperidin-2-one (2.2 g)as an off-white solid. MS (ESI): 282.25 m/z, [M+H]⁺. ¹H NMR (400 MHz,CDCl₃) δ ppm 7.15-7.08 (m, 3H), 4.87 (t, 1H), 3.54-3.49 (m, 1H),3.37-3.34 (m, 1H), 2.51-2.45 (m, 1H), 2.27-2.21 (m, 2H), 2.22-2.10 (m,6H), 1.94 (m, 1H).

Synthesis of 1-(2,6-dimethylphenyl)-3-(pyrrolidin-1-yl) piperidin-2-one

To a solution of 3-bromo-1-(2,6-dimethylphenyl) piperidin-2-one (0.2 g,0.709 mmol) and DIPEA (0.366 g, 2.83 mmol) in acetonitrile (10 mL) wasadded pyrrolidine (0.0655 g, 0.921 mmol) and the resulting reactionmixture was stirred at 90° C. for 16 hours in a sealed tube as progressof the reaction was monitored by TLC (Mobile phase: 50% EtOAc in petether, Visualization: UV). The reaction was concentrated under reducedpressure to afford crude residue which was diluted with water (20 mL)and extracted with ethyl acetate (2×50 mL). The combined organicextracts were dried over anhydrous Na₂SO₄ and concentrated to afford thecrude product which was purified by normal phase flash chromatography(eluted with 25%-30% of EtOAc in Pet ether) to afford1-(2,6-dimethylphenyl)-3-(pyrrolidin-1-yl) piperidin-2-one (350 mg) as apale yellow gummy oil. LCMS purity: 75.17%, MS (ESI): 273.43 m/z,[M+H]⁺.

Synthesis of1-benzyl-1-(1-(2,6-dimethylphenyl)-2-oxopiperidin-3-yl)pyrrolidin-1-iumbromide

To a stirred solution 1-(2,6-dimethylphenyl)-3-(pyrrolidin-1-yl)piperidin-2-one (250 mg, 0.9178 mmol) in acetonitrile (10 mL) was addedwith benzyl bromide (0.2041 g, 1.1931 mmol) and the resulting reactionmixture was stirred at 90° C. for 16 h in a sealed tube as progress ofthe reaction was monitored by TLC (Mobile phase: 10% Methanol in DCM,Visualization: UV). The reaction mixture was cooled to room temperatureand concentrated under reduced pressure to afford the crude productwhich was triturated with EtOAc (20 mL×2) to afford1-benzyl-1-(1-(2,6-dimethylphenyl)-2-oxopiperidin-3-yl) pyrrolidin-1-iumbromide (105 mg) as an off white solid. MS (ESI): m/z 363.4 [M]⁺. ¹H NMR(400 MHz, DMSO-d₆) δ ppm 7.64-7.62 (m, 2H), 7.57-7.48 (m, 3H), 7.20-7.13(m, 3H), 4.78 (m, 3H), 4.03-4.01 (m, 2H), 3.80-3.72 (m, 2H), 3.55-3.50(m, 1H), 3.41-3.61 (m, 1H), 2.51-2.49 (m, 1H), 2.39-2.35 (m, 1H),1.21-1.99 (m, 10H), 1.97-1.90 (m, 2H).

The following examples were prepared from 3-bromo-1-(2,6-dimethylphenyl)piperidin-2-one, benzyl bromide and the appropriate azacycloalkanefollowing procedures described for the synthesis of compound 91A.Products were purified by trituration or reverse phase prep. HPLC.

MS (ESI): Compound # Structure m/z 92A

377.55 [M]⁺ 93A

391.1 [M]⁺

The following examples were prepared from bromo acetyl bromide, benzylbromide and the appropriate azacycloalkane and substituted anilinefollowing procedures to prepare compound 69A.

Compound # Structure MS (ESI): m/z  94A

341.2 [M]⁺  95A

357.2 [M]⁺  96A

348.2 [M]⁺  97A

371.2 [M]⁺  98A

369.3 [M]⁺  99A

385.2 [M]⁺ 100A

349.3 [M]⁺ 101A

345.2 [M]⁺ 102A

359.2 [M]⁺ 103A

373.2 [M]⁺ 104A

337.2 [M]⁺ 105A

351.3 [M]⁺ 106A

355.2 [M]⁺ 107A

371.4 [M]⁺

Example 2—Inhibition of Nav1.7 Current

Representative compounds of the invention were synthesized according tothe described methods and tested for the ability to inhibitvoltage-gated sodium channels.

Manual Patch Clamp:

Cell Culture

NaV1.7 was expressed in HEK293 cells upon induction with tetracycline.Cells were cultured in DMEM containing 10% dialyzed Fetal Bovine Serum(VWR, Radnor, Pa.), 1% Glutamax (VWR, Radnor, Pa.), 1%Penicillin-Streptomycin (VWR, Radnor, Pa.), 100 mg/L Hygromycin (ThermoFisher Scientific, Waltham, Mass. and 5 mg/L Blasticidin (Alfa Aesar,Haverhill, Mass.). Cells were grown and maintained at 37° C. in ahumidified environment containing 10% CO₂ in air. Cells were detachedfrom the culture flask for passage and harvested using 0.05%Trypsin-EDTA (Thermo Fisher Scientific, Waltham, Mass.). To induceNaV1.7, cells were induced with tetracycline (0.1-1 μg/mL, IBIScientific, Peosta, Iowa) the day before recording and plated onto24-well plates. Cells were washed with DPBS (VWR, Radnor, Pa.),trypsinized and then triturated five times in 10 mL of growth media tobreak apart cell aggregates. For one 24-well plate, 2 mL of cellsuspension was mixed with 23 mL of fresh growth media and 0.1-1 μg/mLtetracycline added. 1 ml of mixed media with cells was then added toeach well of a 24-well plate, with a 12 mm coverslip already placed inthe bottom of the well. Cells were then incubated in 37° C. and 10% CO₂overnight.

Patch Clamp Solutions & Drugs

The intracellular solution contained the following (in mM) CsCl 135,NaCl 10, EGTA 10, HEPES 10, MgCl₂ 2, adjusted to pH 7.2 with CsOH. Theexternal solution was a normal Ringer solution containing (in mM) NaCl155, HEPES 10, glucose 10, KCl 3.5, CaCl₂ 1.5, MgCl₂ 1 adjusted to pH7.4 with NaOH. CsCl is from Alfa Aesar, Haverhill, Mass. All otherchemicals are from Sigma-Aldrich, St. Louis, Mo. In order to test thedegree of internal block by test compounds the compounds were dissolvedin internal solution at the indicated test concentration. In controlexperiments the internal solution did not contain any compound. In orderto test the degree of external block by test compounds the compoundswere dissolved in external solution at the indicated test concentration.

Whole Cell Patch Clamp Protocol

18-24 hours after cells were induced with tetracycline, coverslips wereplaced into a chamber filled with Normal Ringer solution at roomtemperature and the chamber placed on a microscope. Pipettes were pulledfrom borosilicate glass on a P97 puller (Sutter Instrument, Novato,Calif.) and polished with a MF-830 Microforge (Narishige InternationalUSA, Inc., Amityville, N.Y.) to have a resistance of 1.5-2.5 MΩ whenfilled with CsCl internal solution at room temperature. Healthy cells(those that are round and translucent with no visible blemishes) werechosen for seal formation. A seal was formed between the pipette and thecell, and a brief pulse of suction was used to “break in” and establishthe whole-cell configuration. The membrane potential was held at −100 mVbefore the voltage protocol began. Only cells with series resistancebetween 1.5-5 MΩ were retained for analysis. The voltage protocol was asfollows: Cells were held at −100 mV for 12 ms followed by ahyperpolarizing step to −105 mV for 12 ms to monitor the leak. Cellswere then stepped back to −100 mV for 40 ms. Cells were then depolarizedto −20 mV for 10 ms and then returned to −100 mV for 26 ms.

Internal Block by Test Compounds

Once the recording was started, the voltage protocol was run at 30second intervals for 5 minutes to get a stable baseline. This wasfollowed by four 30-second periods of 5 Hz stimulation of the samevoltage protocol separated by 1 minute of rest which was then followedby 0.33 Hz stimulation after the last train. Currents were recordedusing PatchMaster software with Heka EPC10 (HEKA Electronics, Lambrecht,Germany). Only cells with inward current amplitudes at −20 mV between400 pA and 4 nA were accepted. In addition, cells having leak currentsgreater than 10% of their current amplitudes were discarded.

Data Analysis: Internal Block

The data was plotted using the Patchmaster software (HEKA Electronics,Lambrecht, Germany) and analyzed by plotting the minimum current duringthe voltage step to −20 mV (peak inward current) as a function of time.In order to determine the degree of rundown over the course of anexperiment, the average peak inward current amplitude (2-3 points)before 5 Hz stimulation was designated as the baseline (I_(baseline)).The average peak inward current during the last 2 second of the last 5Hz train was measured (I_(test)). The control fraction current remainingwas calculated by dividing I_(test) by I_(baseline). On each recordingday three cells were tested with control internal solution and theaverage fraction of current remaining calculated (Ctrl fractioncurrent).

To determine the % block produced by test compounds applied internallythe following was done. The average peak inward current amplitude (2-3points) before 5 Hz stimulation was designated as 0% block(I_(0%block)). To correct for the current change under controlconditions, I_(0%block) was multiplied by the average Ctrl fractioncurrent remaining to get the corrected 0% block current. The averagepeak inward current during the last 2 seconds of the last 5 Hz train wasdesignated as the unblocked current (I_(unblocked)). The % block wascalculated using the following equation:(1−I_(unblocked)/(I_(0%block)*Ctrl fraction current remaining)×100).

Representative examples of the invention were tested for intracellularinhibition of NaV 1.7. Activity Range is % inhibition at 10 μM testconcentration: “+++” (>95%), “+++” 95-70%, “++” (70-40%) or “+” (<400%).The results are presented in the following Table.

Nav1.7 Intracellular Compound Inhibition  1A +++  2A ++  3A ++++  4A +++ 5A +++  6A +++  7A +++  8A +++  9A ++++ 10A +++ 11A ++++ 12A ++++ 13A+++ 14A +++ 15A ++++ 16A ++++ 17A ++++ 18A +++ 19A ++ 20A ++++ 21A ++++22A +++ 23A +++ 24A ++++ 25A ++ 26A +++ 27A +++ 28A ++ 29A +++ 30A ++++31A ++++ 37A +++ 39A +++ 40A ++ 41A +++ 43A +++ 59A +++ 85A ++++ 86A++++

Representative examples of the invention were tested for intracellularinhibition of NaV 1.7. Activity Range is % inhibition at 3 μM testconcentration: “++++” (>90%), “+++” 90-70%, “++” (70-40%) or “+”(<400%). The results are presented in following Table.

Nav1.7 Intracellular Compound Inhibition  3A ++++  4A +++  5A ++++  6A+++  9A +++ 20A ++++ 21A ++++ 22A +++ 24A ++++ 28A + 29A ++++ 30A ++++31A ++++ 32A ++++ 33A ++++ 34A +++ 35A ++++ 36A +++ 37A +++ 38A +++ 44A++++ 45A +++ 46A ++++ 47A ++++ 52A ++++ 53A +++ 54A ++++ 55A +++ 56A ++57A +++ 59A + 62A ++++ 63A ++++ 65A ++++ 66A +++ 68A ++++ 69A ++++ 70A++++ 71A +++ 72A ++++ 73A ++++ 74A +++ 75A ++++ 76A ++++ 77A +++ 80A++++ 81A +++ 82A +++ 85A +++++ 86A ++++ 89A +++ 90A ++++ 91A ++ 93A +++External Block by Test Compounds

Once the recording was started, the voltage protocol was run at 30second intervals for 5 minutes to get a stable baseline. This isfollowed by 5 Hz stimulation of the same voltage protocol run until theend of experiment. The test compound is added during the 5 Hzstimulation train making sure to wait until the cell shows stablecurrent rundown rate before addition of the compound. The test compoundis added for 5 minutes before washing out with normal Ringer's solution.Currents were recorded using PatchMaster software with Heka EPC10 (HEKAElectronics, Lambrecht, Germany). Only cells with inward currentamplitudes at −20 mV between 400 pA and 4 nA were accepted. In addition,cells having leak currents greater than 10% of their current amplitudeswere discarded.

Data Analysis: External Block

The data was plotted using the Patchmaster software (HEKA Electronics,Lambrecht, Germany) and analyzed by plotting the minimum current duringthe voltage step to −20 mV (peak inward current) as a function of time.To determine the % block produced by test compounds applied externallythe following was done. After the stable current rundown rate wasestablished during the 5 Hz stimulation train, the Rate_(rundown) wascalculated by dividing the change in peak current amplitude by time. Theaverage peak inward current amplitude (2-3 seconds) before addition ofcompound was used to determine 0% block (I % block). To correct for therundown, I_(0%block) is subtracted by the (Rate_(rundown)*5 min) to getthe corrected 0% block current. The average peak inward current duringthe last 2-3 seconds of the 5 minutes of compound application timebefore washing is the unblocked current (I_(unblocked)). The % block wasthen calculated using the following equation: Fraction currentblock=1−I_(unblocked)/(I_(0%block)−Rate_(rundown)*5 min).

Representative examples of the invention were tested for extracellularinhibition of NaV 1.7. Activity Range is % inhibition at 10 μM testconcentration: “++++” (>90%), “+++” 90-70%, “++” (70-40%) or “+” (<40%).The results are presented below.

Nav1.7 Extracellular Compound Inhibition  3A ++  4A +  6A +  9A + 33A ++35A + 38A + 44A + 46A ++ 47A ++ 53A + 69A + 70A ++ 76A ++ 80A + 81A +86A + 90A +Automated Patch Clamp:Cell Culture

NaV1.7 was expressed in HEK293 cells upon induction with tetracycline.Cells were cultured in DMEM containing 10% dialyzed Fetal Bovine Serum(VWR, Radnor, Pa.), 1% Glutamax (VWR, Radnor, Pa.), 1%Penicillin-Streptomycin (VWR, Radnor, Pa.), 100 mg/L Hygromycin (ThermoFisher Scientific, Waltham, Mass. and 5 mg/L Blasticidin (Alfa Aesar,Haverhill, Mass.). Cells were grown and maintained at 37° C. in ahumidified environment containing 10% CO₂ in air. Cells were detachedfrom the culture flask for passage and harvested using 0.05%Trypsin-EDTA (Thermo Fisher Scientific, Waltham, Mass.). To induceNaV1.7, cells were induced with tetracycline (0.1-1 μg/mL, IBIScientific, Peosta, Iowa) the day before recording.

Before experiments, cells were washed with DPBS (VWR, Radnor, Pa.),digested with Detachin (VWR Radnor, Pa.) and then triturated 10 times inCHO Serum-Free Media (VWR Radnor, Pa.) to resuspend the cells and tobreak apart cell aggregates. Cells were counted and the finalconcentration was set at 2-5 million cells per mL.

Patch Clamp Solutions & Drugs

The intracellular solution contained the following: 140 mM CsF, 1 mM/5mM EGTA/CsOH, 10 mM HEPES, 10 mM NaCl, adjusted to pH 7.3 with CsOH, andosmolality to 320 with OSM solution. The external solution contained thefollowing: 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl₂), 10 mM HEPES,10 mM Glucose, adjusted to pH 7.4 with CsOH and osmolality to 305 withOSM solution. The OSM solution was a normal Ringer solution containing(in mM) NaCl 155, HEPES 10, sucrose, KCl 3.5, CaCl₂) 1.5, MgCl2 1adjusted to pH 7.4 with NaOH. All chemicals are from Sigma-Aldrich, St.Louis, Mo. In order to test the degree of internal block by testcompounds, the compounds were dissolved in internal solution at theindicated test concentration. In control experiments the internalsolution did not contain any compound. In order to test the degree ofexternal block by test compounds the compounds were dissolved inexternal solution at the indicated test concentration.

Automated Patch Clamp Protocol

Automated Patch Clamps were performed on Qube 384 (Sophion Bioscience,Woburn Mass.) with multihole Qchips at a temperature setting of 22degrees. The whole cells configuration was formed with default Qube sealand break-in parameter. The membrane potential was held at −100 mVbefore the voltage protocol began. Two voltage protocols were followed.

Step 1: Cells were held at −100 mV with a depolarized pulse to −20 mVfor 10 ms, the interval was set at 5 s. Currents were corrected bydefault leak subtraction from every pulse. The duration was set at 5mins.

Step 2: Cells were held at −100 mV with a depolarized pulse to −20 mVfor 10 ms. The frequency was 5 Hz. Currents were corrected by leaksubtraction calculated before step 2. The duration was set at 4 mins.

Internal Block by Test Compounds

After Step 2, the Qchip was removed from the recording chamber. Theinternal solution was changed with solutions containing test compounds.Qchips were held at −100 mV without pulsing after being replaced in therecording chamber. The total solution switching time was 8 minutes.After the internal solution exchange, Cells were recorded and step 2 forrepeated for 10 minutes.

Data analysis was performed by Sophion Analyzer. Cells were filteredwith minimum 50 MOhm seal resistance and minimum 5 nA starting current.The rundown of the currents was corrected with control cells (Non-Drug).The remaining was calculated by averaging last 3 points in the end ofexperiments. The base line was calculated as the average of last 3points of Step 2.

IC₅₀ curves were plotted with DR-plots/Hill function (a dose-responseplot with a Hill fit).

Representative examples of the invention were tested for intracellularinhibition of NaV 1.7 in the automated patch clamp assay. Activity Rangeis reported as IC₅₀: “++++” (<0.3 μM), “+++” (0.3-1 μM), “++” (1-3 μM)or “+” (3-10 μM). The results are presented below.

Nav1.7 Intracellular Compound Inhibition 3 +++ 4 +++ 5 ++ 6 +++ 11  +++14  +++ 20  ++ 23  +++ 29  ++++ 30  ++++ 31  +++ 32A +++ 33A +++ 35A ++36A + 37A +++ 38A ++ 41A + 44A ++++ 45A +++ 46A +++ 47A ++ 48A ++ 49A ++50A ++ 51A + 52A ++++ 53A +++ 54A +++ 55A +++ 56A ++ 57A +++ 58A ++ 60A++ 61A + 62A +++ 63A +++ 64A ++ 65A +++ 66A ++ 67A ++ 68A +++ 69A +++70A +++ 71A +++ 72A +++ 73A +++ 74A +++ 75A ++ 76A ++ 77A +++ 78A + 79A++ 80A +++ 81A +++ 82A +++ 83A +++ 84A + 85A ++ 86A ++ 87A + 88A + 89A++ 90A +++ 91A ++ 92A ++External Block by Test Compounds

After Step 2, the external solution was changed with solutionscontaining test compounds. Qchips were held at −100 mV without pulsing.The total solution switching time was 8 minutes. After the externalsolution exchange, cells were recorded using the same procedure as step2 for 10 minutes.

Data analysis was performed by Sophion Analyzer. Data were correctedwith control cells (Non-Drug). The IC₅₀ data were plotted withDR-plots/Hill function (a dose-response plot with a Hill fit).Representative examples of the invention were tested for extracellularinhibition of NaV 1.7 in the automated patch clamp assay. Activity Rangeis reported as IC₅₀: “++++” (<1 M, “+++” (1-3 μM), “++” (3-10 M) or “+”(>10 μM). The results are presented below.

Nav1.7 Extracellular Compound Inhibition 4 + 5 + 6 + 32A + 35A + 36A +37A + 38A + 41A + 44A + 45A + 46A ++ 47A +++ 49A ++ 51A ++ 53A + 54A +55A + 57A ++ 58A ++ 62A + 63A ++ 64A ++ 66A + 68A + 69A ++ 75A ++ 77A+++ 80A + 81A + 82A ++ 83A +++ 86A + 87A + 88A + 89A + 90A ++ 91A + 92A+

Example 3—Membrane Permeability

The PAMPA assay (pION, Inc., Woburn Mass.) was used to determine theability of compounds of the invention to cross an artificial lipidmembrane by passive diffusion. Test compounds were dissolved in DMSO (10mM) and diluted 200-fold in buffer (pION Inc., pH 7.4) to provide 50 uMstock solutions. Buffer (150 μL) was added to a UV blank plate and stocksolutions (150 μL) were transferred to a UV reference plate. The blankand reference spectrum were read using a spectrophotometer. Stocksolutions (200 μL) were added to the donor plate of the PAMPA sandwichplate and an accept plate painted with GIT lipid (pION Inc, 5) wasplaced on top. Buffer (200 μL) was added to the acceptor plate and thePAMPA sandwich plate was incubated for 4 hours. Aliquots (150 μL) fromthe acceptor plate were added to a UV plate and read as acceptorspectrum. Aliquots (150 μL) of the donor solutions were added to a UVanalysis plate and read as donor spectrum. The permeability coefficientof test compounds was calculated using PAMPA Explorer™ software (version3.5.0.4) based on the AUC of the reference plate, the donor plate, andthe acceptor plate.

The PAMPA permeability results (10⁻⁶ cm/s) of representative compoundsare reported as “+” (<0.1 10⁻⁶ cm/s), “++” (0.1-2.0 10⁻⁶ cm/s), “+++”(2.0-10.010⁻⁶ cm/s) or “++++” (>10.0 10⁻⁶ cm/s).

PAMPA Compound (10⁻⁶ cm/s)  2A +  3A +  5A +  6A +  7A +  8A +  9A +10A + 12A + 13A + 14A + 15A + 16A + 17A + 18A + 19A + 20A + 21A + 23A +26A + 27A + 30A + 31A + 32A + 33A + 34A + 35A + 36A + 37A + 38A + 39A +41A + 42A ++ 44A + 45A + 46A + 47A + 48A + 49A + 50A + 51A + 52A + 53A +54A + 55A +++ 56A + 57A + 58A + 59A + 60A + 61A + 62A + 63A + 64A +65A + 66A + 68A + 69A ++ 71A + 72A + 74A + 75A + 76A + 77A + 78A + 79A +80A + 81A + 82A + 84A + 86A + 89A ++ 90A + 91A + 93A +

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. It will also be understood that noneof the embodiments described herein are mutually exclusive and may becombined in various ways without departing from the scope of theinvention encompassed by the appended claims.

What is claimed is:
 1. A method for treating pain, cough, itch, or aneurogenic inflammatory disorder in a patient, comprising administeringto said patient an effective amount of the compound represented byFormula (I)

wherein: Y⁻ is a pharmaceutically acceptable anion; R^(A), R^(B), andR^(C) are each independently selected from H, D, halogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, OR¹, CN, NR^(J)R^(K),NR^(L)C(O)R^(M), S(O)R^(N), S(O)₂R^(N), SO₂R^(O)R^(P), SO₂NR^(Q)R^(R),SO₃R^(S), CO₂R^(T); C(O)R^(U), and C(O)NR^(V)R^(W); each of R^(I),R^(J), R^(K), R^(L), R^(M), R^(N), R^(O), R^(P), R^(Q), R^(R), R^(S),R^(U), R^(V), and R^(W) is independently selected from H, D, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl; or R^(J) andR^(K) or R^(V) and R^(W) or R^(Q) and R^(R) can be taken together withthe nitrogen to which they are attached to form a substituted orunsubstituted 5, 6, 7, or 8 membered ring; R^(T) is independentlyselected from H, D, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, and substituted or unsubstituted alkynyl; R^(A),R^(B), and/or R^(C) can be taken together with the phenyl ring to whichthey are attached can form a fused bicyclic or tricyclic ring system; X¹is selected from —CR^(X)R^(Y)—, —NR^(Z)C(O)—,—NR^(Z)C(O)CR^(X)R^(Y)—OC(O)—, —SC(O)—, —C(O)NR^(1A)—, —C(O)O—, —(O)CS—,—NR^(1A)S(O)—, —S(O)NR^(1A)—, —NR^(1A)C(O)NR^(1A), —S(O)— and —S(O)₂—;each of R^(X), R^(Y), R^(Z), and R^(1A) is independently selected fromH, D, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, and substituted or unsubstituted alkynyl; each of R^(D) andR^(E) is independently selected from H, D, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, and substituted or unsubstituted cycloalkyl; orR^(D) and R^(E) together with the carbon to which they are attached forma substituted or unsubstituted C₃-C₆ cycloalkyl, substituted orunsubstituted heterocyclic; or R^(D) and R^(Z) together with the carbonand the —N—C(O)— to which they are attached form an optionallysubstituted 5-8-membered lactam; R^(F) and R^(G) together with the N⁺ towhich they are attached form an optionally substituted heterocyclic ringhaving zero, one or more nitrogen atoms in addition to the N+; or, eachof R^(F) and R^(G) is independently selected from substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, andsubstituted or unsubstituted C₃-C₆ cycloalkyl; and R^(H) is aryl orheteroaryl, each optionally substituted with substituted orunsubstituted C₁₋₆ alkyl, alkoxy, C₃-C₆ cycloalkyl, heterocyclyl,phenyl, substituted phenyl, heteroaryl, substituted heteroaryl,hydroxyl, amide, sulfonamide, urea, nitrile, or halogen.
 2. The methodof claim 1, wherein Y⁻ is bromide, chloride, or iodide.
 3. The method ofclaim 1, wherein X¹ is —NHC(O)—.
 4. The method of claim 1, wherein R^(H)is a C₆₋₁₀ aryl or a 5- to 10-membered heteroaryl, each optionallysubstituted with a substituted or unsubstituted C₁-C₆ alkyl, halo,nitrile, hydroxyl, and alkoxy.
 5. The method of claim 1, wherein each ofR^(A) and R^(B) is independently selected from H, halogen, C₁₋₄ alkyl,and NR^(J)R^(K); each of R^(J) and R^(K) is independently selected fromH or C₁₋₄ alkyl; and wherein R^(C) is H, halogen, C₁₋₄ alkyl, orNR^(J)R^(K).
 6. The method of claim 1, wherein R^(A), R^(B), and R^(C)are each independently selected from H, D, halogen, OR^(I), substitutedor unsubstituted C₁-C₄ alkyl, and NR^(J)R^(K); wherein each of R^(I),R^(J) and R^(K) is independently selected from H and substituted orunsubstituted C₁-C₄ alkyl.
 7. The method of claim 1, wherein each ofR^(A) and R^(B) is CH₃, and R^(C) is selected from the group consistingof H, CH₃, halogen, nitrile, methoxy, and ethoxy.
 8. The method of claim1, wherein R¹ is C₁₋₄ alkyl optionally substituted with halogen, oxygen,C₃₋₈ cyclic alkyl, aryl, or heteroaryl, and wherein R^(E) is H, D, orC₁₋₄ alkyl.
 9. The method of claim 1, wherein R^(D) and R^(E) are bothhydrogen.
 10. The method of claim 1, wherein R^(D) is hydrogen and R^(E)is an alkyl.
 11. The method of claim 1, wherein R^(D) and R^(E) aretaken together with the carbon to which they are attached to form aC₃-C₆ cycloalkyl.
 12. The method of claim 1, wherein R^(F) and R^(G)together with the N⁺ to which they are attached form a substituted orunsubstituted five, six, seven-, or eight-membered heterocyclic ring.13. The method of claim 1, wherein each of R^(F) and R^(G) isindependently selected from unsubstituted C₁-C₄ alkyl.
 14. The method ofclaim 1, wherein the compound is selected from the Table below: Compound# Structure  1A

 2A

 3A

 4A

 5A

 6A

 7A

 8A

 9A

10A

11A

12A

13A

14A

15A

16A

17A

18A

19A

20A

21A

22A

23A

24A

25A

26A

27A

28

29

30A

31A


15. The method of claim 1, wherein the compound is selected from theTable below: Compound # Structure  1B

 2B

 3B

 4B

 5B

 6B

 7B

 8B

 9B

10B

11B

12B

13B

14B

15B

16B

17B

18B

19B

20B

21B

22B

23B

24B

25B

26B

27B

30B

31B


16. The method of claim 1, wherein said pain is selected from the groupconsisting of pain due to back and neck pain, lower back pain, cancerpain, gynecological and labor pain, fibromyalgia, arthritis, rheumatoidarthritis, osteoarthritis, rheumatological pains, orthopedic pains,acute and post herpetic neuralgia and other neuropathic pains, sicklecell crises, vulvodynia, peri-anal pain, irritable bowel disease,irritable bowel syndrome, inflammatory bowel disease, oral mucositis,esophagitis, interstitial cystitis, urethritis and other urologicalpains, dental pain, headaches, trigeminal trophic syndrome,erythromelalgia, abdominal wall pain, chronic abdominal wall pain,allergic rhinitis, muscle pain, rectal pain, Levator ani syndrome,proctalgia fugax, hemorrhoid pain, stomach pain, skin ulcers, stomachulcers, burn pain, ophthalmic irritation, conjunctivitis, eye redness,dry eye, dry eye syndrome, complex regional pain syndrome, post-surgicalocular pain, postoperative pain, acute postoperative pain, andprocedural pain.
 17. The method of claim 1, wherein said cough isselected from the group consisting of cough in patients with asthma,COPD, asthma-COPD overlap syndrome (ACOS), interstitial pulmonaryfibrosis (IPF), idiopathic pulmonary fibrosis, post viral cough,post-infection cough, chronic idiopathic cough and lung cancer.
 18. Themethod of claim 1, wherein said itch is selected from the groupconsisting of itch due to pruritus, brachioradial pruritus, chronicidiopathic pruritus, genital/anal pruritus, notalgia paresthetica, scalppruritus, allergic dermatitis, contact dermatitis, atopic dermatitis,hand eczema, poison ivy, infections, parasites, insect bites, pregnancy,metabolic disorders, liver or renal failure, drug reactions, allergicreactions, eczema, genital and anal itch, hemorrhoid itch, and cancer.19. The method of claim 1, wherein said neurogenic inflammatory disorderis selected from the group consisting of allergic inflammation, asthma,chronic cough, conjunctivitis, rhinitis, psoriasis, inflammatory boweldisease, interstitial cystitis, arthritis, colitis, contact dermatitis,diabetes, eczema, cystitis, gastritis, migraine headache, rosacea,sunburn, pancreatitis, chronic rhinosinusitis, traumatic brain injury,polymicrobial sepsis, tendinopathies, chronic urticaria, rheumaticdisease, acute lung injury, exposure to irritants, inhalation ofirritants, pollutants, chemical warfare agents, and atopic dermatitis.